Methods and systems for control of microfluidic devices

ABSTRACT

The present invention provides control methods, control systems, and control software for microfluidic devices that operate by moving discrete micro-droplets through a sequence of determined configurations. Such microfluidic devices are preferably constructed in a hierarchical and modular fashion which is reflected in the preferred structure of the provided methods and systems. In particular, the methods are structured into low-level device component control functions, middle-level actuator control functions, and high-level micro-droplet control functions. Advantageously, a microfluidic device may thereby be instructed to perform an intended reaction or analysis by invoking micro-droplet control function that perform intuitive tasks like measuring, mixing, heating, and so forth. The systems are preferably programmable and capable of accommodating microfluidic devices controlled by low voltages and constructed in standardized configurations. Advantageously, a single control system can thereby control numerous different reactions in numerous different microfluidic devices simply by loading different easily understood micro-droplet programs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and claims the benefit ofpriority to, U.S. application Ser. No. 11/251,188, filed Oct. 13, 2005,which is a continuation of, and claims the benefit of priority to, U.S.application Ser. No. 09/819,105, filed Mar. 28, 2001, which areincorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of microfluidics. Moreparticularly, the present invention is directed to control methods,control systems, and control software for microfluidic devices thatoperate by moving discrete micro-droplets through a sequence ofdetermined configurations.

2. Description of the Related Art

Micro/nano technology devices are known in the art as devices withcomponents on the scale of 1 μm to 100 s of μm that cooperate to performvarious desired functions. In particular, microfluidic devices aremicro/nano technology devices that perform fluid handling functions,which, for example, cooperate to carry out a chemical or biochemicalreaction or analysis.

Most microfluidic devices in the prior art are based on fluid flowingthrough microscale passages and chambers, either continuously or inrelatively large aliquots. Fluid flow is usually initiated andcontrolled by electro-osmotic and electrophoretic forces. See, e.g.,U.S. Pat. No. 5,632,876, issued Apr. 27, 1997 and titled “Apparatus andMethods for Controlling Fluid Flow in Microchannels;” U.S. Pat. No.5,992,820, issued Nov. 30, 1999 and titled “Flow Control inMicrofluidics Devices by Controlled Bubble Formation;” U.S. Pat. No.5,637,469, issued Jun. 10, 1997 and titled “Methods and Apparatus forthe Detection of an Analyte Utilizing Mesoscale Flow Systems;” U.S. Pat.No. 5,800,690, issued Sep. 1, 1998 and titled “Variable Control ofElectroosmotic and/or Electrophoretic Forces Within a Fluid-ContainingStructure Via Electrical Forces;” and U.S. Pat. No. 6,001,231, issuedDec. 14, 1999 and titled “Methods and Systems for Monitoring andControlling Fluid Flow Rates in Microfluidic Systems.”

These devices are relatively disadvantageous because, inter alia, theyrequire larger volumes of reactants by virtue of their flow-baseddesign, and fluid control by electro-osmotic and electrophoretic forcestypically requires relatively large voltages, which may be dangerous andare difficult to generate in small portable control devices. Controldevices for microfluidic devices based on such technologies are larger,at least desktop in size.

More advantageous technologies for microfluidic devices have beendeveloped by one or more of the inventors or the present application andothers. This advantageous technology manipulates very small aliquots offluids (known herein as “micro-droplets”) in microscale passages byrelying largely on pressure and other non-electric forces. These devicesare advantageous in that smaller volumes of reagents are required, andin that non-electric forces can be generated by smaller voltages, of theorder of magnitude output by standard microelectronic components. See,i.e., U.S. Pat. No. 6,057,149, issued May 2, 2000 and titled “MicroscaleDevices And Reactions In Microscale Devices;” U.S. Pat. No. 6,048,734,issued Apr. 11, 2000 and titled “Thermal Microvalves in a Fluid FlowMethod;” and U.S. Pat. No. 6,130,098, issued Oct. 10, 2000.

However, to the knowledge of the inventors, no well-structured controlsystems have been provided for such micro-droplet-based microfluidicdevices that exploits the essential advantages of such devices.

Citation or identification of any reference in this Section or anysection of this application shall not be construed that such referenceis available as prior art to the present invention.

SUMMARY OF THE INVENTION

It is one of the objects of the present invention to overcome thisdeficiency in the art and provide methods and systems for controllingmicro-droplet-based microfluidic devices that exploits their essentialadvantages. Because of the structure and properties of such microfluidicdevices, the methods and systems of this invention can be implemented ina wide range of embodiments, from entirely handheld embodiments tolaboratory embodiments for performing high-throughput reactions andanalyses. Further, because of the structure and properties of suchmicrofluidic device, these methods and systems can be controlled byusers to perform diverse reactions and analysis in a manner similar toprogramming a computer.

Thus, the present invention has for one of its several objects theprovision of programmable control systems and software for what areknown herein as “digital” microfluidic devices. The control systemsprovided, reflecting the design of preferred microfluidic devicesthemselves, have a generally hierarchical design in which detailed andlower-level device control is organized into a smaller number of basiccontrol functions, while overall and higher-level device control isorganized as sequences of the basic function that cause a particulardevice to carry out intended reactions of analyses. The control systemsof the present invention are thereby adaptable to many different typesof digital microfluidic devices and intended reactions; they arescalable to devices of various complexities, simple to program andeconomical.

An embodiment includes a system for controlling the operation of amicrofluidic device having a micropassage for holding a micro-volume ofliquid with a volume between one nano-liter and one micro-liter, areaction chamber, and one or more active components. The system includesa processor that can receive a user request for the microfluidic deviceto perform a reaction program, memory including stored instructionscorresponding to hierarchical control signals that can direct themicrofluidic device to perform the user-requested reaction program, anda programmable digital acquisition unit including: a heater drivercircuit, a temperature sensor driver circuit, and a detection drivercircuit that passes signals to at least one active component that candetect reaction products in the reaction chamber. In some aspects, thedigital acquisition unit generates control signals for the activecomponents responsive to the user-requested reaction program thatcontrol (i) heating the micro-volume of liquid, (ii) detecting atemperature related to the micro-volume of liquid, and (iii) detectingreaction products in the micro-volume of liquid.

An embodiment includes a system for controlling a microfluidic devicehaving a micro-channel that can contain a micro-volume of liquid with avolume between one nano-liter and one micro-liter, a reaction chamber,and an actuator including associated and active components that canoperate in coordination to achieve a desired functionality. The systemincludes memory having stored instructions corresponding to auser-selected reaction program. The memory includes a user levelfunction corresponding to the reaction program, a microdroplet levelfunction corresponding to an operation performed on a micro-volume ofliquid contained within a microfluidic device, an actuator levelfunction corresponding to an actuator operation, and a component levelinstruction directing the generation of a control signal for anindividual component of the microfluidic device. The system includes aninterface that can allow an operator to select a desired reactionprogram for the microfluidic device that corresponds to a user levelfunction having a microdroplet level function, which microdroplet levelfunction includes an actuator level function, and the actuator levelfunction includes a component level function. The system furtherincludes control circuitry that can create and transmit the controlsignal responsive to a component level function for controlling thecomponent of the microfluidic device.

The present includes the embodiments recited above, as well as allcombinations of the embodiments with their particular aspects and withthe particular aspects of other embodiments. The invention furtherincludes sub-combinations of these embodiments and aspects. Thisinvention is also understood to include systems for practicing any ofthe described methods, these systems having the preferred hierarchicalstructures described in the following. This invention also includescombinations and sub-combinations of these systems, for example, a dataacquisition board alone, or in combination with user interface hardware,or in combination with software, or in combination with microfluidicprocessor descriptive data, or so forth. This invention also includescomputer readable media or computer memories storing programs forcarrying out the methods of this invention along with necessarydescriptive and state data.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention may be understood more fully by reference to thefollowing detailed description of the preferred embodiment of thepresent invention, illustrative examples of specific embodiments of theinvention, and the appended figures wherein:

FIG. 1 illustrates an exemplary microfluidic device thermal controlledin a preferred manner;

FIG. 2 illustrates a functional hierarchy of the present invention;

FIGS. 3A-B illustrate preferred control system structure of the presentinvention;

FIGS. 4A-C illustrate controlled heating component functions for apreferred microfluidic processor;

FIGS. 5A-B illustrate pressure generator component functions for apreferred microfluidic processor;

FIGS. 6A-D illustrate micro-valve actuator functions for a preferredmicrofluidic processor;

FIGS. 7A-C illustrate micro-droplet motion functions for a preferredmicrofluidic processor;

FIGS. 8A-D illustrate micro-droplet metering functions for a preferredmicrofluidic processor;

FIGS. 9A-E illustrate micro-droplet mixing functions for a preferredmicrofluidic processor;

FIGS. 10A-E illustrate reaction/analysis functions for a preferredmicrofluidic processor;

FIGS. 11A-B illustrate optic detection actuator functions for apreferred microfluidic processor;

FIG. 12 illustrates an exemplary reaction control function; and

FIG. 13 illustrates an exemplary sample preparation method.

In figures of the same numeric but differing alphabetic designation, forexample, FIG. 5A and FIG. 5B, identical elements are referenced with thesame reference characters.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Preferred Microfluidic Devices

This section generally describes preferred microfluidic devicescontrolled by the systems and methods of the present invention. Thesystems and methods of the present invention control microfluidicdevices that operate in a manner referred to herein as “digital”. Inthis sub-section, the general characteristics of “digital” microfluidicdevices are first described. Subsequently, a more preferred type ofthermally-controlled “digital” microfluidic device is described.

Digital Micro-Fluidic Devices

Microfluidic devices perform chemical or biochemical reactions oranalyses by manipulating fluid reagents in chambers and passages whichare generally sized in cross-section from approximately 10 to 50 μm(micro-meter) up to approximately 100 to 1000 μm, and which are formedon or in a usually flat substrate having linear dimensions fromapproximately 1 mm (centimeter) to approximately 20 cm. A microfluidicdevice may manipulate fluid reactants as they flow through the passagesand chambers of the device, either as continuous flows from inputreservoirs through the device to outlet ports, or as semi-continuousflows of fluid aliquots substantially filling the passages and chambersof the device during operation. Alternatively, preferred microfluidicdevices may manipulate fluid reagents as separate and discretemicro-droplets that are characterized by having lengths that areapproximately an order of magnitude or more smaller than the dimensionsof the device, for example, approximately 1 to 20 mm (millimeter) orless. Thus, during operation the passages and chambers of a preferredmicrofluidic device are largely free of fluid reagents. Micro-dropletsmay have, for example, volumes from approximately 1 nl (nanoliter) toapproximately 1 μl (microliter). For example a micro-droplet of size 10μm by 100 μm by 1 mm has a volume of 1 nl; a 100 μm by 1000 μm by 10 mmmicro-droplet has a volume of 1 μl. Each microfluidic device preferablymanipulates micro-droplets of only a few pre-selected sizes or volumes.

“Digital” microfluidic devices, as this term is used and understoodherein, are of the latter preferred type, maintaining and manipulatingfluid reactants as separate and distinguishable micro-droplets. Thepassages and chambers of a digital microfluidic device have a pluralityof predefined positions so that each micro-droplet either resides at oneof the predefined positions or is moving between these predefinedpositions. The predefined positions, known herein as “stable” positions,are positions to which a micro-droplet can be moved and stablely reside,and are created by configuration and arrangement of passages andchambers, and by the forces generated therein. A micro-droplet mayreside motionless at a stable position even while other micro-dropletsare moving between other stable positions. Stable positions may occur inregions where, for example, a force driving a micro-droplet may be madeto vanish so that a micro-droplet becomes stationary, or where an extraforce is required for transit so that in the absence of such extra forcea micro-droplet will remain stationary, or where forces acting on themicro-droplet balance leaving no net driving force. In contrast, theless preferred type of microfluidic device that manipulates continuousor semi-continuous fluid flows through passages may be considered as an“analog” device.

Reactions in a digital microfluidic device may be specified in terms ofmicro-droplet residence at, and transitions between, stable positions.For example, during motion between stable positions, micro-droplets maybe prepared for an intended reaction or analysis by metering a newmicro-droplet from a fluid reservoir, or by mixing two or more existingmicro-droplets into a single new micro-droplet, or so forth. (“Metering”a micro-droplet is taken herein to mean creating a new micro-droplet ofapproximately known volume or size.) A reaction may occur in amicro-droplet having the necessary composition while it resides at astable position. Optionally, reaction in the stationary micro-dropletcan be excited by, for example, heating, cooling, or radiation, or soforth, or may simply occur with the passage of time.

According to the present invention it is useful, but is not intended tobe limiting, to consider digital microfluidic devices as analogous tofinite-state machines (FSMs). FSMs are well known in the computer artsto have a finite number of states or configurations and to accomplishtheir functions through a series of state transitions from initialstates to final states, each state transition being made in response toa determined input. Analogously, a state or configuration of a digitalmicrofluidic device may be defined by a description of themicro-droplets present in the device and the stable position at whicheach micro-droplet currently resides. The number of these configurationsis finite because a digital microfluidic device manipulates a finitenumber of discrete and distinguishable micro-droplets which reside at afinite number of stable positions. Operation of a digital microfluidicdevice to carry out an intended reaction or analysis may then be definedby a sequence of configurations from an initial configuration to a finalconfiguration. In the initial configuration, the device has pre-loadedreagents in their initial positions and is ready for the loading of anyremaining reagents or samples. Intermediate configurations result frommicro-droplet manipulations that form a micro-droplet with theconstituent reagents necessary for the intended reaction in a stablereaction position. In the final configuration, the intended reactiontakes place in this formed micro-droplet.

Accordingly, control methods and systems of the present invention inpreferred embodiments are structured so that reaction control may bespecified in terms of manipulations of micro-droplets, or equivalentlytransitions of microfluidic device configurations. In this structure,details of actual device control, which are device technology andimplementation dependent, appear only in relation to performingmicro-droplet manipulations, and can otherwise be substantially ignoredfor reaction control. In an important aspect, the present inventionstructures the details of device control for micro-droplet manipulationby incorporating advantageous features, techniques, and principles knownin the computer arts for the control of FSMs. For example, the stablepositions in a digital microfluidic device may be considered as one ormore “registers,” the passages between stable states may be consideredas “combinatorial logic” between the registers. The configuration of adevice may be considered as the occupancy of the “registers” bymicro-droplets, and methods and systems for controlled digitalmicrofluidic devices may be designed according to register-transferdesign paradigms known for similar types of FSMs. As a further example,a digital microfluidic device may be controlled in an overlapped orpipelined manner, wherein the various micro-droplets arrive at theirlocations in a particular configuration at different times, so that theconfiguration is not realized at single time throughout the microfluidicdevice. Such overlap, or pipelining, of configurations may beadvantageous because pipelined control, which breaks a single operationinto a sequence of sub-operations, is known in the computer arts to be amethod of achieving increased processor throughput.

In the more preferred embodiments to be described next, the details ofdevice control make important use of the principles of hierarchicalstructure. It is to be understood, however, that this analogy is notintended to lead to limiting inferences. For example, the controlmethods and systems of this invention are not limited to known FSMcontrol methods and techniques.

Preferred Digital Microfluidic Devices (Processors)

The systems and methods of the present invention may be applied tocontrol general digital microfluidic devices as just described. They arepreferably applied, however, to digital microfluidic devices that havethe following additional properties

(i) predominantly modular and hierarchical structures, and

(ii) of controllability mainly by electrical control signals.

Such preferred digital microfluidic devices, hereinafter calledmicrofluidic “processors” (or simply “processors”), are preferred overgeneral digital microfluidic devices because a wide range of differentprocessors may be flexibly and easily controlled by a singleprogrammable control system implementing modular and hierarchicallystructured control methods. Control of a particular microfluidic processof a particular class is then specified by invoking a high-level controlmodule, which hierarchically encapsulates details of low-level controlfor all microfluidic processors of that particular class.

These two preferred properties of microfluidic processors are nextdescribed in detail. The first property, that a microfluidic processorhas a largely modular and hierarchical construction means herein, first,that a microfluidic processor is constructed from a limited number oftypes of basic functional modules, for example, preferably less thanapproximately 10 module types, or more preferably less thanapproximately 5 module types. In terms of the FSM analogy, these basicfunctional types of modules are analogous to the basic functional typesof electrical circuits, for example, NAND, NOR gates, and flip-flops,out of which FSMs may be constructed. In the following, basic modulesare primarily termed “actuators”. A exemplary set of basic actuatortypes, sufficient for many (but not necessarily all) microfluidicprocessors, includes micro-valve type actuators, pressure generationtype actuators (or other types of force generating actuators),heating/cooling type actuators, actuators for monitoring processorstate, and so forth. Micro-valves can be controlled to close or open aparticular passage, preferably reversibly, to the motion ofmicro-droplets, gases, or other passage contents. Pressure generationactuators can be controlled to create relative gas pressure (or relativevacuum). Heating/cooling actuators can be controlled to performlocalized or generalized heating or cooling. Actuators for statemonitoring can be controlled to provide input that signals micro-dropletposition, local processor temperature, or other parameters. Actuatorsfor optical excitation and detection may also be desirable. For example,radiation may initiate a reaction or monitor reaction products;radiation may also be used to monitor micro-droplet position andcomposition.

Modular and hierarchical construction is also taken to mean thatactuators are in turn hierarchically constructed fromatomically-controllable, discretely-implemented, device-levelcomponents. Atomic or discretely-implemented controllable components arethose device-level controllable components that, in the technology usedfor a particular microfluidic processor, may be directly implemented, orare the simplest controllable components, or may not be decomposed intosimpler controllable components, or so forth. Any particularmicrofluidic-processor technology typically does not have single,unitary, components at the device implementation level for the allactuator types. For example, in the preferred thermally-controllableclass of processors to be described, there is no singleatomically-controllable micro-valve component having a micro-valvefunction available for constructing microfluidic processors. Insteadmicro-valve actuator function is built from several individualcomponents, each of which is atomically controllable and discretelyimplemented and which are arranged together and jointly controlled bythe methods of this invention to have a micro-valve function. This is,of course, similar to electrically-controllable “macro-valves,” whichare also built from a number of unitary mechanical and electricalcomponents which function together to perform the valve function. It isalso analogous to FSMs where, for example, NOR gates cannot be directlyimplemented in many semiconductor fabrication technologies, but rathermust be constructed from an arrangement of gates (made in turn fromtransistors) formed from regions of semiconductor, conductor, andinsulator which can be directly implemented. Accordingly, in anyimplementation technology, each basic actuator type is usuallyconstructed from several lower-level and discretely-implementedcomponents which are arranged and controlled to have the particularactuator function. In other words, actuators are usually hierarchicalconstructs of individual device components available in a particularimplementation technology.

Such substantially hierarchical construction does not rule out certainactuator types which may be constructed from only a single devicecomponent. In some technologies, certain actuator functions may, withoutlimitation, be directly implemented. Nor does it rule out a certainamount of “special purpose” microfluidic functions which may be neededto implement certain limited and specialized functions not describableof the basic and generalized actuator functions. Preferably, specialpurpose function occupies less than 20%, and more preferably less than10%, of the area or device count of a processor.

Substantially modular construction also preferably extends to higherdesign levels. At higher design levels, microfluidic processors arebuilt from sub-assemblies of a limited number of sub-assembly types.Each type of sub-assembly is controlled to perform certain type ofmicro-droplet manipulations; in other words, it is sub-assemblies thatare controlled to cause the transitions between device configurationsnecessary for the intended reaction or analysis. In keeping with theprinciple of hierarchical construction, each sub-assembly type is, inturn, built from a number of individual actuators combined andinterconnected with passages, chambers, ports, and so forth. Generally,the methods of this invention have a hierarchical structured in parallelwith the microfluidic processor technology so that actuators arecontrolled primarily to realize sub-assembly function, and device-levelcomponents are controlled primarily to realize actuator function.

Sub-assembly types are preferably limited to less than approximately 10sub-assemblies, or more preferably to less than approximately 5sub-assemblies. An exemplary set of sub-assembly types provides formetering micro-droplets of determined volumes, for movingmicro-droplets, combining two or more micro-droplets, mixing a possiblyheterogeneous micro-droplet, stimulating (or exciting) a reaction in amicro-droplet, observing and detecting reaction products, and so forth.For example, a metering sub-assembly may use a source of gas pressure topinch off a fluid-filled passage of determined volume from a largerfluid reservoir. A sub-assembly for moving a micro-droplet may use apressure generator actuator to generate mechanical force, gas pressure,to push the micro-droplet. A sub-assembly for combining twomicro-droplets may include two inlet passages converging to a singleoutlet passage, the inlet passages being controlled with micro-valveactuators and being provided with micro-droplet motion actuators. Amicro-droplet mixing sub-assembly may be built from a micro-dropletmotion actuator that causes sufficiently rapid motion to induce laminarmixing. A reaction/analysis sub-assembly may be built from a chamber (ora length of passage) with access controlled by micro-valve actuators,and provided with actuators to stimulate the reaction, for example,through the application of heat or radiation. A sub-assembly fordetecting results of reactions or analyses may, for example, employactuators to sense micro-droplet optical properties. Further examples ofactuators and sub-assemblies will be apparent to one of skill in the artin view of the following description of a specific exemplary digitalmicrofluidic processor.

Largely modular and hierarchical construction is not intended to causeneedless limitations or duplications in microfluidic processor design.For example, although each actuator is usually part of a singlesub-assembly, it may be advantageous and economical for a singleactuator to function as part of two or more sub-assemblies. Similarly,one device-level component may function as part of two or moreactuators. How components or actuators may be employed as parts ofhigher-level functional structures is often technology specific.

The second preferred property of preferred microfluidic processors isthat they are controlled primarily by electrical signals, and to alesser extent by optical signals, with other types of signals, such aspneumatic, hydraulic, mechanical or so forth, being employed rarely ifat all. Control signals are generated by control systems of the presentinvention operating according to the methods of the present invention,and are exchanged with a microfluidic processor being controlled inorder to control the directly-controllable, device-level components, andthereby to realize higher-order actuator and sub-assembly functions.Monitoring signals may be transmitted from a controlled microfluidicprocessor to a control system to reflect the effect of prior controlsignals, such as, for example, whether a specified temperature has beenreached, whether a micro-valve opened or closed as controlled, whether amicro-droplet moved as controlled, and so on. In other words, directdevice control, and thus actuator control and sub-assembly control, isdone on or in the microfluidic processor principally in response toelectrical signals with little or no intervention by external devices.Use of external devices is preferably limited to unavoidable loading ofsamples or reactants not initially present in the processor, or tootherwise interfacing with the external environment.

Preferred electrical control signals are of relatively low voltage(preferably less than 50 V (volt), more preferably less than 25 V, andeven more preferably less than 15 V or 10 V. Control signals sent to amicrofluidic processor from a controller may include, for example,electrical inputs causing internal actuator operation, or optical inputsthat excite or probe reaction products. The electrical inputs may bededicated to individual microfluidic processors or, according to anembodiment of the invention, the electrical inputs may be shared in anarray so as to reduce the number of external contacts. Control signalsreceived from a microfluidic processor may include primarily electricaloutputs for monitoring device state, for example, temperature-monitoringsignals, or micro-droplet-position monitoring signals. Optical signaloutputs may monitor micro-droplet presence, micro-droplet opticalcharacteristics to determine the results of a reaction or analysis, orso forth. Whether optical signals are generated and detected on amicrofluidic processor in response to external electrical controlsignals, or whether optical signals are externally generated anddetected in a controller (also in response to electrical signals) andexchanged optically, for example, over fiber-optic paths with aprocessor, is an implementation matter.

Microfluidic processors may be constructed according to any technologythat allows microfluidic processors to be controlledmicro-droplet-configuration-by-micro-droplet-configuration usingexternal electrical signals. For example, microfluidic processors can beconstructed according to the arts of mechanical and silicon-basednano-technologies. Passages may be etched in glass or silicon; valvesmay include flexible silicon elements actuated by applied voltages;fluids may be moved by moveable nano-elements, or by controlledpressure, either available from an external source or generatedinternally. A single microfluidic device can be constructed in a singletechnology, or may include multiple technologies.

Preferred Micro-Fluidic Processors

Preferred microfluidic processors primarily use thermally-controlledactuators with optical signals for monitoring or detection. Inparticular they are constructed according to a technology that useslocal resistive heating or Peltier-device cooling for control functions.For example, a thermally-controlled processor can be maintained atbaseline temperature by a temperature-controlled heat sink or a coolingelement, such as a Peltier device, with actuators controlled bylocalized heating above the baseline. Localized heating may preferablybe provided by low power resistive heaters of less than approximately 1to 2 W, advantageously controlled by low voltages, for example, lessthan 50, 25, 15 or 10 V.

Mechanical force, where needed for control purposes, may be provided bygas pressure generated by localized heating applied to a gas reservoirwithin a processor. For example, controlled gas pressure may be directlyused to cause micro-droplet motion. Controlled gas pressure may also beused to control micro-valves by causing an obstructing element to moveinto and close a passage, while return to normal pressure may draw theobstructing element back and open the passage. In a preferredembodiment, the obstructing element may be a low melting point solid,which is melted for valve operation also by localized heating.Thermally-controlled micro-valves may, act to admit externally providedrelative pressure or relative vacuum into a processor for powering morecomplex actuators. Thermally-controlled mechanical force may also begenerated by other means, such as by other heat-sensitive fluids, bydifferentially expandable materials, and so forth. Additionally,localized heating and cooling may be directly applied to micro-dropletsfor reaction control. Further, electrical signals may be used foractuator control in other manners, such as attractive or repulsivemagnetic or electric forces.

In this embodiment, device monitoring signals are derived primarily fromtemperature sensitive elements mounted in the device, which preferablygenerate electrical monitoring signals such as, for example,temperature-sensitive resistive or semiconductor elements. Localizedheating may be precisely controlled by sensed temperatures. Gaspressures may then be controlled by controlled localized heating. Localthermal capacity may be monitored by a combination of a temperaturesensor with a small heater by measuring temperature responses withrespect to a determined quantity of heat. Using local thermal capacitysensors, micro-droplet presence or absence may be sensed because amicro-droplet has a higher thermal capacity than an otherwise emptypassage. Other electrical monitoring signals may be generated by, forexample, detecting local electrical impedance, which may providealternative means for detecting micro-droplet presence. Micro-sensorswith deformable conductive elements may provide for direct detection oflocal pressures.

Optical signals may be used in preferred microfluidic processors whereadvantageous. For example, scattered radiation may provide the simplestmeans of detecting or observing reaction or analysis results. Incidentradiation may be helpful to initiate or stimulate a reaction oranalysis. Also, micro-droplet position sensors may be optically based.

In more detail, FIG. 1 illustrates, schematically and not to scale, anexemplary integrated microfluidic processor constructed in the preferredmodular and hierarchical manner in an embodiment of the preferredthermal-control technology. This integrated microfluidic processor isdesigned to perform a sample analysis through the following steps: meterpre-determined micro-droplets from two sources, for example, a source ofthe sample and a source of analysis reagents; mix the meteredmicro-droplets to form a third homogeneous micro-droplet; perform atemperature-controlled analysis reaction in the third micro-droplet; andfinally, optically monitor the analysis results.

This exemplary microfluidic processor is constructed from three types ofsub-assemblies, each sub-assembly being constructed from three types ofactuators, and each actuator being constructed from one type ofcontrollable device-level component. The processor also contains passivecomponents such as passages, reservoirs, ports, outlets, opticconductors, and so forth. In particular, this processor has fourseparate sub-assemblies: two micro-droplet metering sub-assemblies,metering1 and metering2; one mixing Sub-assembly, mixing 1; and onereaction/detection sub-assembly; referenced reaction/detection1. Thesesub-assemblies are constructed from three controllable heater actuators,six controllable valve actuators, and one optical detector, allinterconnected with passive inlets, overflows, vents, and reservoirs.The sub-assemblies have the following components: sub-assembly metering1includes inlet1, overflow1, valve1, heater1, and passage 1; sub-assemblymetering2 includes inlet2, overflow2, valve2, heater2, and passage 2;sub-assembly mixing1 includes heater 1 (and optionally heater2), valve3,valve4, vent1, vent2, Y-shaped passage 3 and passage 4; and sub-assemblyreaction/detection1 includes valves5, valve6, heater3, and passage 5.Here, heater1 and heater2 are included in both the mixing and in themetering sub-assemblies. Also, heater 1, valve3, valve4, vent1, vent2,and passages 1 and 4 alone may form a micro-droplet motion sub-assembly.Lastly, in addition to passive passages, the processor is constructedfrom only one type of controllable device-level components, localizedresistive heaters. Preferably, resistive heaters are operatively coupledto resistive temperature detectors that provide feedback information.

Before a description of sub-assembly operation, exemplary passageconfigurations for creating and defining stable positions are described.Generally, stable positions are created by hydrophobic regions, or bythe relative arrangement of main passages and vented side passages.(Main passages are continuing passages along which micro-droplets aremanipulated; side passage are dead-end passages branching from the mainpassages.) First, hydrophobic regions, for example regions h1-h6 of FIG.1, are limited regions whose interiors have been treated to assume ahydrophobic character, whereas the remainder of the passage interiorshave a hydrophilic, or at least a wettable character (either normally orby treatment). Because of surface tension effects in micro-droplets,predominantly aqueous micro-droplets will travel in the hydrophilicregions of passages with smaller hindrance than when they travel in thehydrophobic regions. In effect, therefore, a barrier exists at junctionsbetween hydrophilic and hydrophobic regions: the hydrophilic regions“attract” aqueous micro-droplets, while the hydrophobic regions “repel”such micro-droplets. Thus, these hydrophobic-hydrophilic junctionsdefine relatively stable positions that a micro-droplet requires extraforce to traverse. Because of the “repulsive” effects of the hydrophobicentrance regions h1, h2, h5, and h6 of the passages to heater1, heater2,vent1, and vent2 in FIG. 1, in comparison with the “attractive” effectsof the substantially hydrophillic interiors of adjacent passages 1, 2,and 4, aqueous micro-droplets are restrained from penetrating into thesehydrophobically-“protected” passages. Similarly, extra force is requiredto cause aqueous micro-droplets to pass the hydrophobically-protectedregions h3 and h4, which therefore define stable regions between mainpassages 1-2 and Y-shaped main passage 3. In the case of predominantlyhydrophobic micro-droplets, the hydrophobic and hydrophilic passagecharacteristics are reversed.

The present invention includes other methods of creating stablepositions that will be apparent to one of skill in the art in view ofthe present description. For example, by placing a controllable ventadjacent to a passage with a valve, a stable position may be createdwhen the valve is closed and the vent is open.

Because the effect of gravitational forces is negligible at the spatialdimensions used in these devices, surface tension may be exploited bydesigning local passage size differences within the device, perhaps inconjunction with adjacent hydrophobic regions. For example, since anarrowed passage will draw fluid from a larger passage by the capillaryeffects of surface tension, a relatively stable position can be createdwhere a relatively narrow passage joins a relatively wider passage. Thisstable position may be reinforced by the presence of an adjacenthydrophobic region.

Stable positions can also be created by a local configuration ofpassages, preferably where a hydrophobically-protected side passage isvented to the exterior branches from a main passage. For example in FIG.1, if a micro-droplet is being moved along passage 4 toward vent 3 bypressure applied to its left surface, and if valve3 is closed whilevalve4 is open, then the micro-droplet will come to reside at the stableposition in passage 5 just beyond the entrance to the side passageleading to vent2. The micro-droplet will not penetrate the side passageto vent2 because of hydrophobic region h6, and it will not pass intopassage 5 because all applied pressure will be released through vent2 tothe exterior. Therefore this position, just beyond the side passage tovent2, is a stable position if valve3 and valve4 are properly actuated.(If valve4 is closed, the micro-droplet will continue moving throughpassage 5.) In this manner valved and vented side passages withhydrophobically-protected entrances also define stable positions.

In summary, hydrophibic regions h3 and h4 create adjacent stablepositions in passages 1 and 2, respectively. Side passages to vent1 andvent2, hydrophobically-protected by regions h5 and h6, respectively,define stable regions adjacent and to the right of their junctions withpassage 4.

Now turning to actuator and then to sub-assembly operations, micro-valveactuators, for example, valve1-valve6, preferably use meltable elements,for example, m1-m6, respectively, to reversibly obstruct, under thecontrol of gas pressure, their respective controlled passages. Forsimplicity of illustration only, micro-valves are schematicallyillustrated FIG. 1 as having only one heater element, whereas, in apreferred subsequently-described embodiment (FIGS. 6A-B), they usuallyhave three separate heaters and one temperature sensor (also up to threetemperature sensors). Heater1 and heater2, which heat their respectivelygas reservoirs, form thermally-controlled gas pressure generatoractuators, which are part of micro-droplet motion and formationsub-assemblies. Heater 3, which heats passage 5, provides for thermalcontrol of reactions in micro-droplets present in this passage. Resultsof reactions completed in passage 5 are detected in this exemplarymicrofluidic processor by an optical actuator, namely input opticconductor o1, which conducts incident radiation to the reaction region,and output optic conductor o2, which conducts scattered and emittedradiation from the sample for analysis. The incident radiation may be inthe IR, visible, or UV bands as required for a particular application.Other detection means can be employed in other applications.

Operations of the sub-assemblies result from the coordinated operationsof their component actuators. First, two micro-droplet motion actuatorsmove micro-droplets along passages 1 and 2 by means of gas pressuresgenerated by pressure generators controlled by heater1 and heater2,respectively. Next, sub-assembly metering1, which is composed ofactuators valve1, heater1, inlet1, overflow1, and passage 1, meters amicro-droplet of determined volume from an aliquot of fluid introducedthrough port inlet1 in the following manner. Initially, if not alreadyopen, valve3 and valve 1 are opened so that the side passage to vent1 isnot blocked. Next, fluid introduced into inlet1, for example, by anexternal manual or robotic device, and flows up to the stable positioncreated by the first hydrophobic region h3 just beyond the widening ofpassage 1, with any excess fluid flowing out through port overfolow1.Region h1 prevents the introduced fluid from entering the side passageto heater1. Finally, controlled gas pressure generated by heater 1pinches the micro-droplet from the introduced fluid that lies betweenthe junction of the side passage to heater1 and region h3, and propelsit to just beyond the junction with the side passage to vent 1. Regionh5 prevents the micro-droplet from entering the side passage to vent1,and vent 1 allows the propelling gas pressure to escape. Sub-assemblymetering2 is constructed and operates similarly. (Optionally, valves,not illustrated, may be present adjacent to inlet 1 and inlet2 in orderto prevent passages 1 and 2 to refill after droplet metering.)

Sub-assembly mixing1 mixes two micro-droplets of differing constituents,which have been adjacently positioned at the stable position created bythe junction of main passage 4 and the side passage to vent1, in thefollowing manner. First, valve3 (and valve1 and valve2) are closed sothat the adjacently situated micro-droplets in passage 4 can bepropelled toward passage 5. Next, gas pressure is generated by heater 1,or by heater2, or by both, so that the two micro-droplets in passage 4are moved to the stable position just beyond the junction of the sidepassage to vent2. Importantly, the generated pressure is controlled sothat the motion is sufficiently rapid to mix the micro-droplets.Finally, the remaining sub-assembly illustrated in FIG. 1, sub-assemblyreaction/detection1, which includes valve5, valve6, heater2, o1, o2, andpassage 5, operates as follows. After a mixed micro-droplet of thecorrect composition is positioned in passage 5, this passage is sealedby closing valve5 and valve6. Next, heater3 is controlled to stimulate areaction in the trapped micro-droplet, and the results of the stimulatedreaction are optically detected by radiation conducted by o1 and o2.

FIG. 1 also illustrates leads and external connectors for the electricaland optical signals. For example, control and monitoring leads 8 forvalve 1 are schematically illustrated as two leads extending from thevalve to the microfluidic processor's edge terminating in connectors 10.(A full and complete illustration of a micro-valve preferably has four,or six or more signal leads.) Although leads 8 are illustrated here assubstantially straight, in most microfluidic processors with moreactuators and leads, leads bend to avoid obstacles and other leads, orare combined where control requirements allow, or crossover each otherseparated by insulating films. The terminating connectors are preferablystandardized, for example, as an array of pins that may be accommodatedby an external socket, or, illustrated here, as rounded protrusionsalong processor edges that may be accepted by mating contacts in areceptacle in a control system. Also, exemplary optic conductors o1 ando2 are illustrated as extending substantially straight from thereaction/detection sub-assembly to optical couplings or connectors 7,also preferably standardized for routine connection to externalradiation sources and detectors. Also, these conductors may need to bendor crossover obstacles. Optical conductors may comprise light pipes,optical fibers, or other means for spatial transmission of an opticalsignal.

According to a preferred embodiment of the invention, the number ofterminating connectors required for the control of a plurality ofactuators may be reduced by arranging/sharing, in the form of an array,the contact wiring to each actuator. The resulting compression of thenumber of terminating connectors advantageously simplifies communicationwith the entire microfluidic processor. Whereas each actuator requirestwo leads to complete an electrical circuit, according to a conventionalarrangement of leads and contacts, a device comprising N actuatorscomprises 2N leads and 2N terminal contacts. By configuring the contactwiring in an array, however, the number of required terminal connectorscan be reduced to as few as 2 N. For example, in a hypothetical devicecomprising 100 actuators, the number of external contacts can be reducedfrom 200 to 20. This greatly simplifies external wiring and devicecontrol.

As stated above, the compression is accomplished by arranging thecontacts in an array. According to this arrangement, electrical contactsfor the N actuators are configured in R rows and C columns such that theproduct RC=N, preferably where R is approximately equal to C, and mostpreferably where R=C. With this arrangement, actuators located in agiven row share a common electrical contact. Similarly, actuatorsarranged in a given column also share a contact. Each actuator has aunique address, though, given by its unique row/column combination.Therefore, each actuator is individually actuatable by supplyingelectric current to the appropriate row-column combination.

It is also preferable that microfluidic processors for control by thepresent invention be physically standardized so that microfluidicprocessors designed for different reactions or analyses may becontrolled by a single external control systems. Standardization would,for example, limit a microfluidic processor to only a few selectedsizes. Electrical and optical connectors would be limited to standardforms, positions, and alignments. Inlet ports, overflow ports, vents,and so forth would be limited to standard forms and locations (for easyrobotic access). A further preferable feature of microfluidic processorsthat promotes standardization is a self-description function. Aprocessor may be described by providing its controllable and passivecomponents, their mutual relations and interconnections, and, for eachcontrollable component, the identity of the connectors for its controland monitoring signals. This self-descriptive information may be used bythe control methods and systems to generate correct control signals atcorrect connectors for a particular microfluidic processor, otherwisesuch self-descriptive information must be explicitly entered by a useror “hard-coded” into the methods. This function may be variouslyimplemented. For example, all the self-descriptive information may bestored in the microfluidic processors; alternatively, a processor maystore a key to a database of this self-descriptive information which isstored elsewhere.

Further description of the construction and functioning of preferredmicrofluidic processors are provided in U.S. Pat. Nos. 6,048,734,6,057,149, and 6,130,098, issued Apr. 11, 2000, May 2, 2000, and Oct.10, 2000, respectively. These patents are incorporated herein in theirentireties by reference without any admission that they are prior art tothe inventions claimed herein.

Preferred Control Systems and Method

This section describes preferred embodiments of these control systemsand methods in view of the characteristics of preferred microfluidicdevices. The control systems of the present invention control generaldigital microfluidic devices, and generate physical control informationin proper modalities and sequences for causing the microfluidic devicesto perform an intended reaction or analysis as a sequence ofconfigurations or “state” transitions. Starting from an initialconfiguration, the microfluidic device is controlled to pass through aseries of intermediary configurations, and to complete operation at afinal configuration in which the intended reaction or analysis isperformed. Each sequential configuration transition typically resultsfrom the creation of a new micro-droplet such as by metering, mixing, ormoving a micro-droplet; the excitation of a micro-droplet by thermal oroptical means, the detection of reaction results, and so forth. Duringthese operations, a microfluidic device preferably generates monitoringsignals that the control systems and methods use to insure successfulcompletion of each operation.

In preferred embodiments, the control systems and methods of thisinvention control digital microfluidic devices that are also modularlyand hierarchically constructed and controlled with electrical andoptical signals as described above. In other words, in preferredembodiments the present invention controls microfluidic processors. Morepreferably, controlled microfluidic processors are implemented in athermally-controlled technology and are physically standardized, also asdescribed. Although the following description is largely limited to thismore preferred embodiment, one of skill in the art will readilyappreciate how to generalize the preferred embodiments described for thecontrol of general microfluidic processors of other technologies, andalso of general digital microfluidic devices.

Therefore, in this more preferred embodiment, the control systems of thepresent invention generate electrical (and optical) signals forcontrolling the individually-controllable device-level components ofpreferred thermally-controlled microfluidic processors. Optionally, thesystems also receive electrical (and optical) monitoring signals. Then,the control methods of this invention command the control systems of thepresent invention to generate signals that reflect the modular andhierarchical construction of the preferred microfluidic processors.Signals controlling the individually-controllable device-levelcomponents are generated to cause these components to function togetheras actuators. Further, these signals are generated to cause theactuators to function together as sub-assemblies that manipulatemicro-droplets. At the highest control level, these signals aregenerated to cause the sub-assemblies to function together so that themicrofluidic processor as a whole performs an intended reaction oranalysis, preferably by passing through a sequence of predeterminedconfigurations designed to perform the reaction.

This hierarchical signal generation can also be considered ashierarchically constrained signal generation. To repeat, at the devicelevel, a preferred microfluidic processor is composed ofindividually-controllable components that may be constructed accordingto a chosen nano-technology as a single atomic and elementary entity,not substantially or significantly decomposable for construction as agroup of more elemental entities. The first constraint is that controlsignals are generated so that these individually-controllable componentsfunction together only as actuators, that is so that the componentcontrol signals are determined by requested actuator control functions.A second constraint is then that the actuator control functions aregenerated so that the separate, controllable actuators act together onlyas sub-assemblies manipulating processing, that is so that actuatorcontrol functions are determined by requested sub-assembly controlfunction, also perhaps represented as sub-assembly control signals.Finally, the “sub-assembly control” functions are requested according toa script, or program, so that the microfluidic processor performsconfigurations leading from an initial configuration to a configurationin which the intended reaction or analysis is performed.

From either viewpoint, a microfluidic processor is preferably controlledaccording to the present invention by “programming” the control systemso that the sub-assemblies function to achieve the correctconfigurations in the correct sequence necessary to complete a reaction.This “programming” is in terms of sub-assembly function, such as thecreation, mixing, movement, thermal (or other) excitation, and detectionof reaction results in micro-droplets. In other words, this“programming” is in terms, intuitively familiar from the chemistrylaboratory where reagent are measured, mixed, heated, and so forth. Itis the systems and methods of the present invention that then convert,or interpret, or otherwise process such a “sub-assembly program” togenerate the correct detailed control signals for all theindividually-controllable microfluidic processor components, andpreferably generate these signals so that the individual microfluidicprocessor components function as actuators and that the actuatorsfunction as sub-assemblies. Stated differently, these methods of thepresent invention, performed by the systems of the present invention,enforce the described hierarchical constraints and encapsulate thedevice-level detail of a controlled microfluidic processor. The end useris presented with a vastly simplified control, or “programming” task.

These “sub-assembly programs” are performed by control systems of thepresent invention which are preferably structured as an at leasttwo-level hierarchy. At the highest level are one or more programmablecomponents, for example, a PC-type computer or an embeddedmicroprocessor. At the next level is include peripheral interfacecircuitry which is under control of the programmable components andwhich actually generates and responds to the electrical (and optical)control signals. The methods of the present invention are thenpreferably implemented as programs for this programmable apparatus thatcause the programmable apparatus to control the peripheral circuitry togenerate and receive control signals passed to and from theindividually-controllable components in the controlled microfluidicprocessor.

In more detail, the “sub-assembly” programs, which are supplied by auser to cause a microfluidic processor to perform an intended reactionor analysis, are alternatively lists of sub-assembly functions that theprocessor is caused to perform in order, or lists of processorconfigurations that the processor is caused to assume in order.Optionally, these program lists may include commands for testing,branching, or iteration. Testing and branching are advantageous where amicrofluidic processor generates monitoring signals and where thesystems and methods of this invention make the monitored informationavailable at the “sub-assembly program” level. The methods of thepresent invention then convert, compile, interpret, or otherwise causethe programmable control systems acting through the peripheral controlcircuitry to generate the requested hierarchically structured orconstrained control signals for the microfluidic processor.

In a preferred implementation, the hierarchical structure of controlsignals, or, equivalently, the constraints on control signal generation,may then be preferably implemented as a corresponding hierarchicalstructure of signal generation functions. In such a structure, whenfunctions at a particular level act in a correctly constrained manneronly by means of functions of lower levels, signal structure andconstraints will be automatically and easily maintained at all levels aslong as the lower-level functions also maintain their lower-levelconstraints. For example, “sub-assembly-level” functions performmicro-droplet functions by requesting correct sequences of“actuator-level” functions without concern for how actuators areimplemented by individual processor components. “Actuator-level”functions request correct sequences of device commands, without concernfor how a thermally-controlled microfluidic processor is implemented.Finally, only “component-level” functions actually convert user programsto control signals generation and actually receive monitoring signals,and contain most details of thermally-controlled device implementation.

Such a hierarchical organization of control functions, along withattendant data, may be expressed in many suitable programming languagesand software engineering paradigms. On one hand, the methods of thepresent invention may wait to translate user-provided, sub-assemblyprograms into function requests until operation of the controlledmicrofluidic processor. For example, component, actuator, andsub-assembly control functions may be implemented as objects in anobject-oriented programing system (using an object-oriented languagesuch as C++). Here, the control functions are object methods and areexecuted in sequence in response to method message exchanged duringoperation. Similarly, the methods may be implemented as an interpretivesystem which also invokes functions only during operation. On the otherhand, these methods may translate programs during an initial compilationstep. For example, the control functions of the various levels may beimplemented as macros (using a procedural language with a macro facilitysuch as C) in a procedural paradigm, which translate each sub-assemblycommand into a corresponding plurality of actuator commands, so thatprograms are translated into instructions for the programmableapparatus. Mixed implementations are possible. For example, controlfunctions can be represented as library routines, or higher-levelfunctions may be objects and lower-level functions may be macros.

Data for the methods of the present invention includes, for example, thecurrent configuration of the microfluidic processor and the currentstate of the actuators and components in the processor. These data can(including micro-droplet changes between successive configurations) berepresented in manners advantageously suited to use by micro-dropletcontrol functions.

Control Methods

This sub-section describes preferred structures for the control signalgeneration functions along with preferred structures for their data andparameters, both for a thermally-controlled microfluidic processor ofthe preferred implementation. The following descriptions apply to anyimplementation paradigm: for implementation with objects, the objecthierarchy is described; for procedural implementation with macros, themacro inclusion hierarchy is described; for procedural implementationwith library routines, the procedure invocation hierarchy is described.One of skill in the art will be readily able to apply the followingdescription to the chosen paradigm. Also, although the followingdescribes a currently preferred allocation of functions to hierarchicallevels, the methods of this invention are readily adaptable to otherfunction allocations, and even to other function definitions. Inparticular, the grouping of components into actuators may beimplementation- and technology-dependent. Also, there may be fewerfunctional levels, for example, just sub-assembly and actuator levels,or more functional levels, where advantageous.

Preferred Functional Structures

FIG. 2 illustrates an exemplary and non-limiting, but preferred,hierarchical organization of signal generation functions for athermally-controlled microfluidic processor implemented in a preferredtechnology. This figure illustrates four function levels, a componentlevel, an actuator level, a sub-assembly (which is functionallyidentified in FIG. 2 as a configuration or micro-droplet level), and auser level. Since higher-level function act only by invoking lower-levelfunctions, they necessarily abide not only by their own constraints butalso by the constraints of all lower-level functions. As described, thisinsures that processor control signals ultimately generated abide by theentire preferred hierarchical structure and constraints.

First, the lowest-level functions are component-level functions 15 b,which are preferably the only functions that directly cause generationof electrical and optical signals for control of theindividually-controllable microfluidic processor components. Forexample, the “current/voltage control” primitive function causes thecontrol system to generate or monitor specified electrical controlsignals. The “external switch control” function causes the controlsystem to switch these signal generators to signal connectors. (The“internal switch control” function controls switches internal to amicrofluidic processor, which, if present, route electrical controlsignals from processor to connectors to internal components.) Therefore,the joint action of these two functions generates and monitorselectrical control signals between the control system andelectrically-controlled processor components. Correct connectors forcomponent control of particular components may be determined from thepreviously-described self-descriptive microfluidic processor data, whichincludes such connector-component information. In the case of preferredself-descriptive microfluidic processors, this self-descriptiveinformation, or a key to it, can be obtained from the microfluidicprocessor itself. The function reading this information from a processoris called the “sense device type” function. Finally, the functions“laser diode control” and “photodiode control” provide similar controlof optical signals.

Level 15 may also include certain additional simple functions 15 a,which implement actions somewhat more complex than the actions ofatomically implementable device components, but which are neverthelesssimple and best classified as components rather than as actuators.Functions 15 a may invoke functions 15 b or other functions 15 a. Anexample of such a generalized component-level function is the “sensetemperature” function, which outputs the temperature at a given sensorelement. Given a specified (resistive) temperature monitor element, itsexternal contacts may be indicated by descriptive microfluidic processordata. The electrical output of these indicated contacts may then bemonitored by the “current/voltage control” and “external switch control”functions, and then converted to a temperature in view of known physicalproperties of the given sensor. The “controlled heating” may apply,using the more primitive control and switch functions, a given power toa given heater element, or may adjust the applied power in view of theoutput of a “sense temperature” function to achieve a given temperature.

These component-level functions and their suggested implementations arenot intended to be limiting. First, other and additional component-levelfunctions may be defined; the listed functions are exemplary and notexhaustive. Second, since component-level functions are typicallydetermined by the implementation technologies, they typically willdiffer for microfluidic processors of different technology. Even withina single technology, details of heating, sensing, and so forth differ indifferent specific microfluidic processor implementations. Further, evenfor a single processor type, different preferred embodiments may packagethe primitive and generalized component-level functions differently.

Actuator-level 16 includes functions that control groups of one or moreusually interconnected components in a manner and sequence so that theyfunction together to achieve a particular actuator-type function.Actuator-type functions are those typically associated with the“plumbing” or “machinery” necessary to implement a chemical reaction,such as opening or closing a micro-valve in the microfluidic processor,generating pressure, sensing quantities, and so forth. For example, a“sense reaction results” function may be optically implemented. It mayact by means of the “laser diode control” and the “photodiode control”functions, first, to cause the proper incident radiation to be providedto the proper external optical connectors in order that reaction resultsare illuminated, and second, to cause scattered or emitted radiation tobe observed. A “sense micro-droplet” function may sense the presence orabsence of a micro-droplet by, in effect, measuring a local thermalcapacity. Thus, this actuator function may, first, provide a givenquantity of heat by means of the “controlled heating” function, andsecond, determine the temperature response by means of the “sensetemperature” function. A greater temperature increase indicates a lowerheat capacity indicative of the absence of a micro-droplet, and viceversa. This function may also be optically implemented to determinemicro-droplet presence or absence in a region by sensing opticalproperties of the region in a manner similar to the “sense reactionresults” function. A “generate pressure” function may use the“controlled heating” function at a given power or to a given temperaturein order to heat gas in a reservoir to increased pressure. Generatedpressure may be monitored with a pressure sensor if available in themicrofluidic processor. Finally, the important “open/close” valvefunctions are described subsequently.

Information describing an actuator's individual components andinterconnection indexed by an actuator identifier may be available fromself-descriptive processor data. In this case, simply an actuatoridentifier may be specified to the actuator functions, which thenautomatically determine component parts from the self-descriptiveprocessor data without requiring user attention or input of thisinformation. In turn, component information, for example, connectoridentification, may be automatically determined by component-levelfunctions from this same data.

Actuator-type functions are expected to be more standardized thancomponent-level functions because they reflect facilities needed byvirtually all microfluidic reaction processors. For example, virtuallyall microfluidic processors will have micro-valves with open and closevalve functions. Nevertheless, the actuator-level functions and theirsuggested implementations described herein are exemplary, and notexhaustive or intended to be limiting. For example, certaincomponent-level functions, especially generalized functions 15 a, may beconsidered actuator functions in different implementations. Second, eventhough many of these types of actuator functions may be substantiallysimilar in different processors, their implementation may differ fromthose suggested above depending on the processor components available inthe implementation technology. Third, different actuator functions maybe present to take advantage of different component types present ondifferent processors, for example, a wider range of sensing actuatorsmay be present to take advantage of a broader sensing technology.

Configuration/micro-droplet 17 functions (performed, generally, bysub-assemblies) are those that act on micro-droplets, preferablyinvoking primarily actuator functions 16 so that micro-droplets movefrom stable position to stable position. Therefore, theconfiguration/micro-droplet 17 functions provide that the microfluidicprocessor progresses through configurations that are defined by themicro-droplets present in a processor and their stable positions. Inother words, a micro-droplet function starts with one or moremicro-droplets at stable positions and invokes actuator functions sothat upon completion the one or more micro-droplets are again atdifferent stable positions. These functions do not complete withmicro-droplets at unstable positions, positions from which amicro-droplet may spontaneously move and in an indeterminate manner.Micro-droplets at unstable positions would therefore make predictableand orderly operation of a microfluidic processor impossible, and thissituation is to be avoided.

Input information for micro-droplet functions includes positions of themicro-droplets to be acted on. Preferably, this information may beobtained from an initial processor configuration, which is updated withnew micro-droplet positions, to a final configuration upon functiontermination. Also where sense actuators are present, these functions maycheck micro-droplet position and report an error if the measuredposition and intended position are inconsistent. Even more preferably,using micro-droplet position and processor self-descriptive data, thesefunctions automatically determine which actuators to invoke forachieving the intended result. Otherwise, micro-droplet position, andpossibly also the correct actuators, must be determined by a user(assuming prior micro-droplet operations were successful) and then inputto these functions.

Micro-droplet-level functions are preferably provided to correspond tostandard types of chemistry laboratory operations, such as measuring,mixing, heating, and so forth. Thus, functions 17 usually include:functions to meter a micro-droplet from a fluid source in order to forma new micro-droplet of known volume, to move a micro-droplet from onestable position to another stable position, to mix an inhomogeneousmicro-droplet to form a homogeneous micro-droplet, to perform a reactionby thermal or other type of excitation, and so forth.

Because microfluidic processors of this invention act in a digitalmanner by manipulating micro-droplets to perform chemical or biologicalanalysis, basic micro-droplet functions types are largely “microfluidicprocessor independent.” Certain micro-droplet functions, for example,separation of micro-droplet constituents, may be added where required bya certain type of reaction. Alternatively, certain combinations of basicmicro-droplet functions may be made available as a single function forefficiency. Variation in function details and function implementationmay occur between different technologies and processor types. Preferredimplementations of these functions for preferred processors aresubsequently described.

User-level 18 functions do the work useful to an end user, performingand monitoring an intended reaction or analysis in a microfluidicprocessor. Functions 18 a, “protocol/compiler/interpreter” functions,direct a microfluidic processor to actually carry out an intendedreaction. These key functions interpret, convert, compile, or otherwiseprocess a user-provided reaction program, preferably specifiedsubstantially as a sequence of micro-droplet-level functions thatprepare a micro-droplet containing the necessary reactants, cause theintended reaction to occur in this prepared micro-droplet, and thendetect or sense reaction results. As described, reactions are preferably“programmed” largely by invoking micro-droplet-level functions, and relyon the function hierarchy of this invention to ultimately generate thenecessary control signals on the correct connectors to cause amicrofluidic processor to perform the invoked functions. Becausemicro-droplet functions, as well as actuator and component function,encapsulate most details of processor actuator operation, users mayadvantageously specify reactions in terms corresponding to routinechemical laboratory operations. Self-descriptive microfluidic processordata permits this specification without attention to internal processordetails.

User-level 18 may also contain operator-type functions 18 b, whichprovide for microfluidic processor control by permitting the selectionof the reaction or analysis “program” to be performed by a microfluidicprocessor, by initiating the selected reaction “program” after readyingthe processor, and by terminating the reaction and returning the sensedreaction results, and so forth. Operator function may also provide formonitoring a microfluidic processor as it processes a reaction. Forexample, monitoring functions may show on an appropriate display devicea graphical (or otherwise formatted) portrayal of the current state of amicrofluidic processor such as the current position of micro-droplets,the current state of microfluidic processor actuators and components,and so forth, along with indications of the “program” steps alreadyperformed and yet to be performed. Optionally, operator-type functionsmay include program development and debugging tools, for example, toolsfor entering micro-droplet function commands, for “single-stepping” aprocessor through a program, and for further facilities familiar fromprogramming environments for computer systems. Since a function of aparticular hierarchical level performs its actions by making requests offunctions, the exchange of requests is fundamental and is variouslyreferred to herein. For example, a higher-level function may generate,or send, or transmit, or so forth a request, which a lower-levelfunction then processes, or accepts, or receives, or so forth.Alternatively, a higher-level function may provide a request to alower-level function.

Preferred Data Structures

The hierarchically arranged signal-generating functions preferablyutilize and maintain certain data, for example, self-descriptive datafor the microfluidic processor, data descriptive of the currentprocessor state, and the configuration or state of micro-dropletspresent in the processor. Self-descriptive data for a microfluidicprocessor generally specifies the processor's components, how they areinterconnected, and by what external contacts they are controlled. Forexample, processor components may be described as a list of atomiccomponents, their type, properties, and where controllable, controlconnectors. Actuators may be also described as a list of their type,properties, and atomic components out of which they are constructed. Theexternal contacts controlling the components of an actuator can bedetermined from the component of the actuator and the connectorscontrolling these components. Component interconnection may be describedby a list of the passages, hydrophobic regions, inlet ports, outletports, vents, and so forth, along with indications of the connectivityof these elements, which may be represented as a network flow diagram.

The self-descriptive processor data may be automatically supplied,preferably by the microfluidic processor, or less preferably by thecontrol system or by both acting in combination. In one embodiment, aROM-type memory (or EPROM, or other permanent or quasi-permanent memory)is embedded in or on a microfluidic processor containing at least thisprocessor-descriptive data. Alternatively, this memory can be limited toa few (≦10) bytes that store only key-type information for lookup in acontrol system database retrieving complete self-descriptive data. Inanother embodiment, machine-readable indicia, such as a bar code, orhuman-readable indicia, such as a serial number, may be provided on amicrofluidic processor. The “sense device type” component functionobtains this self-descriptive data either by accessing the embeddedmicrofluidic processor memory by means of standardized connectors (forexample connections “1, 2, 3, and 4” on all microfluidic processors), orby reading machine-readable indicia, or by manual input ofhuman-readable indicia.

Self-descriptive microfluidic processor data preferably permitssimplified parameterization of the component and actuator-levelfunctions by the symbolically identified components and actuators. Forexample, a “controlled heating” function may be applied to “heater-6B”,wherein “heater-6B” is identified by the functions in theself-descriptive data. In contrast, applying a “controlled heating”function to external contacts 39, 42, 43, and 68 is less flexible. An“open/close valve” function may be more preferably applied to“valve-12”, instead of to “valve-12”'s components or to theirconnectors. Information describing a microfluidic processor alsopreferably includes the state of the symbolically-identified componentsand actuators. For example, the current temperature, or the past heatingof “heater-6B” is 80 C; valve-12” is currently “open”; and so forth.

Function data further includes micro-droplet configuration or “state”data, which includes a list of the micro-droplets currently present in amicrofluidic processor and their composition and current position.Micro-droplet composition may, for example, be recorded by the source orsources from which the micro-droplet was created. Micro-droplet positionrecords its current unstable position occurring only transiently duringtransitions between configurations. Micro-droplets may be symbolicallyspecified in the configuration, for example, the sixth micro-dropletcreated being “micro-droplet-6,” and micro-droplet functions may then beapplied to symbolically specified micro-droplets. For example, when the“move micro-droplet” function is applied to “micro-droplet-6” thefunction determines this micro-droplet's current position from thecurrent processor configuration. From this determined position, the“move micro-droplet” function next determines from the self-descriptiveprocessor data the correct actuators to invoke to move“micro-droplet-6,” and from current state information, the current stateof these actuators. When the determined actuators are invoked theircomponents, their component's connectors, and their component's stateare similarly determined. Alternatively, in simpler but less preferredembodiments, the actuators, components, and connectors may bepre-specified.

Finally, user-level operator monitoring and display functions maydisplay this function data. For example, animation of microfluidicprocessor operation may be displayed as a map of the microfluidicprocessor components and their connections along with the currentposition of the micro-droplet and the current component activations.Limited aspects of the current state may also be operator-selected fordisplay.

In one embodiment of the present invention, a microfluidic processor maybe represented in an object-oriented programming paradigm. In anexemplary object representation, where some or all of the components,actuators, micro-droplets, and so forth may be represented as objects,the maintained data would be represented as object instance data,defining for each object its type, state, geometric relation to otherobjects, and so forth. The control functions would be methodsmanipulating the component, actuator, and micro-droplet objects. Thesemicrofluidic processor control function may be represented in otherprogramming paradigms where the maintained data may be represented aslists, tables, trees, or other known data structures.

Control Systems

A control system of the present invention preferably has a distributedand hierarchical structure, generally paralleling the hierarchicalcontrol function structure illustrated in FIG. 2. Preferably,lowest-level control functions, such as component-level functions 15 andactuator-level functions 16, are implemented in system interfacehardware configured for direct connection to a controlled microfluidicprocessor (for example, data acquisition and control board 26 in FIG.3A), while highest-level functions, user-level functions 18, especiallyoperator functions 18 b, are implemented in system user hardwareconfigured for user interaction (for example, personal computer 27 inFIG. 3A). Intermediate function levels, reaction control 18 a level,micro-droplet level 17 (or configuration level), and actuator level 16may be implemented in the interface or in the user hardware, or in anintermediary hardware level, as convenient. (Micro-droplet level 17functions are those functions performed by the physical sub-assembliesdescribed above, which in turn are composed of actuators and perhapsindividual components.)

Control systems, and especially system interface hardware, may beimplemented with electronic microprocessor, such as those available fromIntel, Motorola or other electronic suppliers. To avoid confusion, suchcontrol system electronic processors will be always called“microprocessors,” while microfluidic processors will be called both“microfluidic processors” and simply “processors.”

FIG. 3A illustrates an exemplary preferred two-level control.Microfluidic processor 20 is illustrated as having a standardizedphysical configuration including a standardized size, shape, andelectrical and optical connectors 21, which are arranged along threeedges of the rectangular processor. The processor is shown beinginserted into (or removed from) an interface hardware receptacle havingelectrical and optical connectors 25 standardized to mate with contacts21 of the processor. Most connectors are for electrical signals, whilecertain are for optical signals (IR, visible, UV) in the case ofoptically monitored or excited microfluidic processors. Further,exemplary microfluidic processor 20 is illustrated with three inletports 22 for accepting fluid reagents or samples. Preferably, theseinlet ports are in standard position on the processor so that laboratoryrobot 24, where available, may be easily programmed for automaticloading of ports of several types of microfluidic processors. Otherwise,the ports should be accessible for manual loading. Where possible,reagents may also be pre-packaged on a microfluidic processor.Additionally, processor 20 has micro-circuit 23 accessible throughcertain standard connectors for storing at least self-descriptiveprocessor information. Alternately, processor 20 may bear indicia, suchas a bar code, indicating device type or further information.

Illustrated first-level, interface hardware comprises data acquisition(“DAQ”) board 26 directly connected to microfluidic processor 20. Apreferred DAQ board is programmable, for example including an embeddedmicroprocessor (such as those produced by Intel, Motorola, etc.) withRAM memory (for example, 1-8 MB), which controls electrical and opticalsensor/driver circuits and switches between outputs of these circuitsand connectors 25. The sensor/driver circuits are switched amongconnectors 25 under microprocessor control to provide control signals tothe microfluidic processor, or to receive monitoring signals. Opticalsignaling components, for example laser diode radiation sources andphotodiode radiation detectors, are similarly controlled by themicroprocessor. The DAQ board also preferably includes a standardizedexternal interface that permit links to a broad range of higher-levelportions of the control system. Illustrated here is generic 5-wire,bi-directional, serial interface 28, similar to such standard interfacesas UART, USB, Firewire, Ethernet, and so forth, all of which may be usedin this invention. In other embodiments, the DAQ board can be configuredto plug into the busses of higher-level control systems. User hardwarepreferably communicates with a DAQ by means of message exchangeaccording to a standard protocol.

A DAQ board with sufficient microprocessor and memory resources mayperform virtually all control functions. For example, such a board mayperform component-level functions 15, actuator-level functions 16,micro-droplet-level functions 17, and reaction-control function 18 a. Inthis preferred embodiment, only the user interface functions are moreefficiently performed on user hardware. Such a capable DAQ board wouldfunction with most user hardware of limited resources. With a lesscapable DAQ-board, control functions may be advantageously shifted touser hardware, starting with higher-level reaction control functions andproceeding lower in the function hierarchy. In the former case, limitedmonitoring messages would need to be exchanged between the DAQ board andthe user hardware; in the latter case, user hardware would sendparameterized messages to the DAQ board invoking lower-level functions.These messages may be divided into packets for actual transfer acrossthe DAQ interface, and the transfer may be error checked.

In alternative embodiments, certain lowest-level control functions maybe offloaded from the DAQ board onto control hardware embedded inprocessor 20 itself, for example, onto micro-circuit 23. For example,this circuit could serve as an internal switch so that a smaller numberof external contacts 21 may be switched among a larger number of controlor monitoring leads on the processor, thus conserving external contacts.Other certain component control functions may be offloaded to themicrofluidic processor.

User hardware (also called herein a “host”) is the top-level of thecontrol systems of this invention. In most embodiments user hardwareperforms at least user interface functions 18 b in FIG. 2. In responseto user input, these top-level functions have final control of starting,monitoring, and stopping a reaction on a processor, and of reportingreaction results. The user hardware, or host, further may performadministrative functions, among which may be managing the softwareinstructions and data for itself and for attached DAQ boards. Softwareinstructions for causing the host to perform its functions may be loadedfrom computer-readable media, such as optical disk 29, or may bedownloaded from network interconnection 30. Data may also be loaded tothe host computer from computer readable media, in particular a databaseof microfluidic processor descriptive data may be loaded into the host.Further, the host may “download” software instructions and data to theDAQ board, where such is not already resident by being stored in, forexample, a ROM/Flash memory card or a small hard disk. This downloadedsoftware and data loaded causes the DAQ board to perform its assignedtasks. User hardware is preferably programmable, for example, withmicroprocessor, memory, and storage, and connects to a controlled DAQboard by means of the standardized interface on the DAQ board.

The hierarchical control systems of this invention—the user hardware,the DAQ board and, optionally, the microfluidic processor itself—mayconveniently be constructed to a number of different design pointssuitable for different applications. As illustrated in FIG. 3A, userhardware 27 may be a laptop PC, typically with a microprocessor of 500Mhz or greater speed, with 64 MB or more of memory, and connected tostand-alone DAQ board 26 by bi-directional UART 28 which plugs into thePC. This implementation is suitable for portable medium-throughputapplications or for light in-laboratory use.

A still more portable design point is a handheld analysis system, inwhich host 27 may be a palmtop or other type of handheld type computer,DAQ board 26 plugs into an “expansion” socket or other receptacle orplug on the handheld host, and microfluidic processor 20 in turn plugsinto a DAQ board receptacle. The handheld may also include remotecommunication interfaces, such as wireless access. This design pointwould have medical applications in a doctor's office, or at bedside, orin an emergency situation, or so forth. It may also have industrialapplications for the “field” of manufacturing processes of industrialchemicals. Other applications will be readily apparent to those of skillin the art.

Another design point is a less portable, but higher throughputlaboratory analysis system in which host 27 may be any PC-type orworkstation-type laboratory computer and one or more DAQ boards 26 withmicrofluidic processors 20 arranged in a number of appropriateconfigurations. In a simple arrangement, the DAQ board may reside in atabletop holder (not illustrated) which connects to host 27 via datacable 28. Alternatively, multiple microfluidic processors 20 with theirassociated DAQ boards may reside in a single holder, or multipleholders, and may be connected to host 27 by a network connections suchas Ethernet connections. For more complete laboratory automation, one ormore processors 20 with their associated DAQ boards may be arranged sothat samples or reactants may be introduced into the processors by oneor more standard laboratory robots. In FIG. 3A this is illustrated bylaboratory robot 24 which has access to inlet ports 22 of microfluidicprocessor 20. This laboratory robot is controlled via cable 31 from host27 so that microfluidic processor loading and processor operation can beconveniently and automatically controlled from a single computer.Alternatively, the robot may be controlled by a separate computer.

A broad range of further design points that are suitable for variousother applications will be apparent to those of skill in the art.

Preferred Thermally-Controlled Embodiment

This section describes more preferred thermally-controlled microfluidicdevices and their more preferred control systems and methods;furthermore, this section describes additional embodiments.

In a more preferred embodiment, this systems and methods of thisinvention are applied to thermally-controlled microfluidic processors,such as is illustrated in FIG. 1. This sub-section describes in orderthe systems and the methods of this more preferred embodiment.

DAQ Board Architecture

The DAQ board of this embodiment is relatively more capable, andtherefore may by interfaced to user equipment, or hosts, of a wide rangeof capabilities. DAQ board architecture includes both a preferredhardware architecture and a preferred system software architecture,described herein.

Hardware Architecture

FIG. 3B illustrates a preferred hardware architecture for DAQ boards ofthis embodiment. First, DAQ boards have one or more receptacles, slots,sockets, or so forth where one or more replaceable microfluidicprocessors may be accommodated in a firmly supporting manner with goodcontact to its external connectors. Microfluidic processors, arepreferably mounted on a relatively tough substrate, for example a PCBboard. Processor substrates are standardized to have one, or at most afew, selected shapes, sizes, and connector arrangements for easyreplacement in one, or at most a few, corresponding DAQ board receptacletypes. Thus, FIG. 3B illustrates microfluidic processor 37 mounted onsubstrate 36.

Standardized electrical connectors 38 a connect between both electricalcontrol lines 39 and lines on substrate 36 leading to microfluidicprocessor 37, and also between electrical monitoring lines 40 andcorresponding substrate lines. Optical connectors 38 b connect betweenboth optical conductors 42 from DAQ board light sources and opticalconductors 41 to DAQ board light sensors and corresponding opticalconductors on processor substrate 36 also leading to the microfluidicprocessor. The electrical connectors, which may have many embodiments,are illustrated here as edge connectors that are engaged when theprocessor substrate is inserted in a DAQ board receptacle.Alternatively, connectors may be suitable for engaging a flexible ribboncable, or may by multi-pin sockets, or so forth. The optical connectorsmay be of types known for connecting fiber-optic cables.

Host computer interface 44 is preferably selected according to the typeof host used in a particular control system. For example, for handheldhosts the DAQ board may plug into an available slot or interfaceintegrated into the handheld device. For laboratory systems using PC orworkstation type hosts, the DAQ board provides a modular, simple, andpreferably standardized connector and interface, for example, suitablefor a USB, or a Firewire, or an Ethernet cable connection. Illustratedin FIG. 3B is a simple, bi-directional, UART serial interface with cableconnector 38 c. The illustrated interface has serial data-in anddata-out lines and a reset line, which should be capable of bringing theDAQ board to a known state. This interface also provides power andground lines.

The DAQ board is preferably externally powered by a host computer (or bya standalone holder). Power may be supplied at standard voltages, forexample, at +12 V, +5 V, or other voltage, which the board itselfconverts to and regulates at required internal voltages. Preferably, aDAQ board is able to negotiate with host (or with its holder) concerningthe power requirements of the board and an attached microfluidicprocessor, and to generate an error indication if the power supply doesnot meet requirements. Similar power negotiations are known from USBinterfaces employed in personal computers.

FIG. 3B generally illustrates a preferred microprocessor-based DAQ boardarchitecture. Microprocessor and memory 43 (such as RAM or ROM)communicate with both host interface controller 44 and with internal buscontroller 45 over a microprocessor bus optimized for high speedcommunication with a few devices. Internal bus 46 is typically differentfrom the microprocessor bus because it is designed and optimized forcontrolling and monitoring interfaces to numerous, lower-speedperipheral circuit controllers. Internal bus controller 45 links themicroprocessor bus bi-directionally with the internal bus.Alternatively, the microprocessor bus may directly connect to peripheralcircuit controllers, and the internal bus may be eliminated. Althoughnot illustrated, the DAQ board may also include one or more hard disksof small form factor, readers for flash devices, RAM, or otherinterfaces.

In an economical embodiment, the signal generation and sensing functionincludes peripheral circuitry in which a smaller number ofbus-controlled signal generation and monitoring circuits are switched(or multiplexed) by bus-controlled signal switching circuits among alarger number of leads or lines for connection to a microfluidicprocessor. Thus, the microprocessor controls microfluidic processorcontrol-signals by controlling the signal generation and signalswitching circuits by means of the internal bus 46. Alternatively, adriver/sensor circuit may be provided for each external connector, andthe signal switches may be eliminated.

Accordingly, FIG. 3B illustrates heater driver 47 circuit, controlled bybus 46, with relatively few outputs leads being switched or routed byanalog switch 48, also controlled by bus 46, among relatively morenumerous control lines 39. The heater driver circuits may control heaterelements on the microfluidic processor by providing either a source ofconstant voltage or current, or a source of pulses of controlled widthor frequency, or of sources of signals of other modulation schemes.Heater elements should be controllable from zero power up to a maximumpower, where the maximum is preferably from 1.0 to 2.0 W, and morepreferably from 0.5 to 2.5 W. Microfluidic processors also typicallyhave at least one cooling device, for example a Peltier device, which isused to establish a baseline operating temperature appropriate to thereaction or analysis being performed, for example, a room temperature ofapproximately 25° C. or lower. DAQ boards, therefore, also includeperipheral circuitry, controllable by the microprocessor for controllingsuch a cooling device.

Similarly, monitoring signals generated in a microfluidic processor andconducted on relatively more numerous monitoring lines 40 are switchedby switch 50, under control of bus 46, to one of relatively fewer numberof digital sensor circuits 49, which may be an analog-to-digitalconverter of similar. Also, the sensor circuits may also provide signalsto activate sensors where needed. The digitized monitoring signals arethen transmitted to microprocessor and memory 43 over internal bus 46.Monitoring signals are typically generated by temperature detectors,preferably at least one detector accompanying and for control of eachresistive heater. Temperature detectors are preferably resistivetemperature detectors (preferably of platinum) with resistance in therange of 100Ω to 4000Ω at 25° C. Since temperature measurementspreferably have an accuracy and resolution of approximately 0.5° C.,temperature sensor circuitry should be able to measure a resistance (forplatinum temperature detectors) in the above range with an accuracy andresolution of better than approximately 0.25%, and more preferablybetter than approximately 0.13%.

Similar switch-based control may be used for optical signals. FIG. 3Billustrates bus-controlled analog switch 54 which switches abus-provided control signal to relatively numerous laser diodes anddrivers 53. Laser diode output is then conducted by light conductors 42to substrate 36, and then to microfluidic processor 37. To provideexcitation light to a microfluidic processor, a DAQ board has at leastone, and preferably two or more, laser diodes (or other controllablelight sources) with a power range of 1-10 mW and with wavelengths usefulfor reaction excitation and detection. Preferably, a plurality of laserdiodes are provided with a plurality of wavelengths specific toplurality of different microfluidic processors performing a plurality ofdifferent reactions or analyses. Further, the laser diodes, or theiroptic conductors, may optionally be provided with optic elements, suchas filters or polarizers. Driver circuits for the laser diodes arepreferably controllable (by the microprocessor) so that laser diodeoutput power can be adjusted over their range.

Optical monitoring signals are received over light conductors 41 and aresensed by photodiodes 51 (or other light sensors). The digitizedphotodiode output is switched onto the bus by switches 54. To monitorlight returned from a microfluidic processor, a DAQ board preferably hasone or more photodiodes, preferably four, or five, or more photodiodeswith characteristics, such as wavelength responsiveness, dark current,quantum efficiency, and so forth, specific to the reactions or analysis.Preferably, a plurality of photodiodes are provided with a variety ofcharacteristics specific to a variety of microfluidic processorsperforming a variety of reactions or analyses. Further, the photodiodes,or the optic conductors, may optionally be provided with optic elements,such as spectral filters, to adapt their responsiveness to the reaction.Photodiode digitization circuits preferably have adjustable gains andranges to accommodate photodiodes of different characteristics.

Alternatively, where controllable optical switches are economicallyavailable, this architecture illustrated may be replaced by a switchedarchitecture similar to that for electrical signal generation andmonitoring, namely fewer optical sources and sensors optically switchedamong more numerous optical conductors.

FIG. 3B is intended to illustrate, not limit, the preferred DAQ boardarchitecture. First, this architecture is easily scalable. Sincemicrofluidic processors typically have numerous electrically drivenheaters and electrical sensors many of which may operate in parallel, aDAQ board preferably has capability to simultaneously drive at least twoheaters and to simultaneously sense at least two monitoring leads by,for example, having two or more analog switch/driver or analogswitch/sensor pairs. Although simultaneous generation and monitoring ofmore than one optical signal is usually not required, this capabilitymay be provided if necessary in the case of electrical signals. Second,DAQ boards may be based on other types of programmable devices and mayhave other arrangement of components for generating control signals andsensing monitoring signals that will be apparent to one of skill in theart in view of the above description. For example, the internal bus maybe eliminated in favor of direct communication between themicroprocessor and the signal generation/monitoring elements. Also, oneor more switches may be eliminated in favor of an increased number ofsignal generation or sensing circuits. Finally, a single DAQ board mayhave receptacles and peripheral circuitry for controlling more than onemicrofluidic processor.

Software Architecture

Software instruction executed by microprocessor 43 (or otherprogrammable control element) controls the DAQ board. In particular,responses to host messages and control signal generation are enabledaccording to the hierarchical microfluidic processor control functions.Although allocation of control system function among a host, a DAQboard, and a microfluidic processor is flexible, preferably, asdescribed, the DAQ board performs most of the control functions in orderthat the microfluidic processor need provide only self-identificationand in order that the user equipment need only provide an operatorinterface. Thereby, microfluidic processor cost is reduced, and the userequipment is freed from real-time microfluidic processor control.

A preferred software architecture is layered as is known in the art. Atthe lowest layer is an “operating system”, which preferably provides,for example, standard software process control, communication, memoryallocation, and access for control of DAQ-board peripheral circuitry.Software process and memory control preferably provides real-time,asynchronous control with interfaces for standard languages, such as Cor C++. Drivers for peripheral circuitry preferably provide asynchronouscontrol over the electrical and optical signals output to a microfluidicprocessor and asynchronous sensing of monitoring signals from acontrolled processor. Such a system may be built, for example, from aminimal Linux kernel augmented with peripheral circuitry drivers.

In a software process-based method implementation, the operating systemexecutes software processes managing, for example, microfluidicprocessor control functions, host communication, and internal DAQ boardadministrative functions. Host communication software processespreferably implement a layered communication protocols. At a networklayer, communication is preferably packet based with error checking (forexample, by a checksum with retransmission of lost or corrupt packets).At a physical layer, the protocol may be implemented over hostcommunication link, such as the illustrated serial link from hostinterface 44, Ethernet, or so forth with provision for negotiation oftransmission rates, packet sizes, and so forth. Exemplary protocols maybe selected from the IP family, such as SLIP or TCP, or from other knownprotocols.

Internal administrative software processes provide responses to, forexample, host requests for DAQ-board status, and for the operation andstatus of an attached microfluidic processor. Administrative softwareprocesses may also provide for DAQ-board software update. For example,in response to a host status request, the DAQ board should report itsstatus (e.g., free, reaction in progress, steps completed, results nowavailable, and so forth). The DAQ board may also perform diagnostictests of the board itself and calibrate on-board sensor circuitry. Inresponse to a power requirements request, the DAQ board should negotiatethe power it expects to draw from the host in advance (e.g. for thisparticular reaction in this particular microfluidic processor). Inresponse to a software update request, the DAQ board should request oraccept software (or firmware) from the host. Further internal statusrequests and responses may also be provided for.

Microfluidic processor control software processes perform functions thathave been generally described with respect to FIG. 2 above, and will bedescribed in more detail below for the preferred thermally-controlledmicrofluidic processors. In a preferred embodiment, component-level,actuator-level, micro-droplet-level, and the user-provided reactioncontrol function are performed by DAQ board software processes.Preferably, at least, functions for drop metering and mixing,temperature cycling, and separation of micro-droplet components in aseparation media are performed on a DAQ board. In a softwareprocess-based embodiment, functions for the software control processesare hierarchically structured as are the function themselves. Forexample, an actuator software process sends request messages to itscomponent-level software processes. Other control implementations willbe apparent to those of skill in the art.

Methods and Functions

This sub-section describes control function for preferredthermally-controlled microfluidic processors, component-level functions,actuator-level functions, micro-droplet-level functions, and lastlyuser-level functions. This description is exemplary and not limiting. Inview of the following description, one of skill in the art willunderstand how to construct other implementations of the describedfunctions, and also how other possible components and actuators, whichmay constructed in the preferred thermally-controlled technologies, maybe controlled according to this invention.

Temperature Control Functions

Temperature sensing and controlled heating are important component-levelfunctions for preferred thermally-controlled microfluidic processors.Temperature sensor elements are preferably resistive elements (resistivetemperature detectors or “RTDs”) configured to have measurableresistance changes in response to temperature changes. Such a sensor maybe made of platinum with resistance in the range of approximately 1000Ω(Ohm) to 4000Ω at 25° C., so that an accuracy and resolution ofapproximately 0.5° C. can be achieved with sensor circuitry capable ofresistance measurements of approximately 0.25% or better accuracy andresolution.

FIG. 4A illustrates an exemplary RTD, which can operate in at least twomodes. FIG. 4B illustrates a function performing the first mode oftemperature measurement. The function first obtains input parameters,here principally the identity of the particular RTD in question. RTDidentity may be provided, for example, as an input to a proceduralfunction invocation, or can be a local variable in an objectrepresenting this RTD, or by other means. However provided, thisidentity determines the control leads (and thus the DAQ-boardconnectors) to be used for measurement, for example, leads 57-60 in FIG.4A, so that the DAQ-board microprocessor can control the appropriateperipheral circuitry. Next, a small current is applied across the RTD onone pair of leads, for example, leads 57 and 60, while the resultingvoltage is sensed across a second pair of leads, for example, leads 58and 59. Finally, the resistance of the RTD is determined from thesupplied current and measured voltage (or vice versa), and thetemperature is then converted from the measured resistance. The appliedcurrent is chosen small enough not to generate significant localheating, but large enough to generate a voltage drop measurable at theabove precision. The use of two pairs of leads improves accuracy,because, since the voltage measurement can be made with little to nocurrent, little or no voltage drop develops in measurement leads 58 and59; most voltage drop measured being measured across the RTD itself.Alternatively, where less accuracy is sufficient, a single pair of leadscan be used for current supply and voltage measurement.

In a second mode, the RTD can sense the presence or absence of amicro-droplet by measuring a local specific heat, which is greater whena micro-droplet is present in a nearby passage nearby than when nomicro-droplet is present. This mode functions in a manner substantiallysimilar to the first mode except that the applied current is greater andis applied for a time sufficient to generate enough heat to increase thesurrounding temperature by a measurable amount, for example, byapproximately, 2° to 4° C. in the absence of a micro-droplet. In thepresence of a micro-droplet, the temperature increase will be less.Therefore, the presence or absence of a micro-droplet can be sensed bymeasuring the rate of the temperature increase.

Heaters are also preferably resistive and configured to controllablygenerate between 0.5 and 1.5 W of heat with a low voltage source. Sincea preferred low source voltage is 5-10 V or less, the resistance of theresistive heaters in the range of approximately 15Ω to 1000Ω at 25° C.(even smaller heaters may be needed for source voltages of less than 5V). As FIG. 4A illustrates, a heater with a nearby RTD may provide forcontrolled heating.

FIG. 4C illustrates a component-level controlled heating function. Inputparameters include the identity of the heater/RTD pair, so that themicroprocessor via the internal bus can energize or monitor the correctleads (and thus the correct DAQ-board connectors), and a desiredtemperature and temperature tolerance. Using a temperature sensingfunction, for example, the function illustrated in FIG. 4B, thetemperature at the heater is determined. The heater current is thenadjusted in view of the measured temperature, the desired temperature,and the tolerance. The time delay is chosen to provide for smoothcontrol characteristics. These control steps, especially the currentadjustment step, may also implement an alternative control method, suchas a PID or a fuzzy logic method, that may depend on the currentlymeasured temperature and on one or more temperatures measured in therecent past.

A further temperature-related, component-level function provides forcontrolling baseline device temperature. In addition to heaters, apreferred microfluidic processor may have a Peltier (or other) coolingdevice (or devices) in order to generally maintain the processor at abaseline temperature, for example, at a room temperature of 25° C.Alternatively, a Peltier cooler can be mounted on the DAQ board in amanner such that it makes thermal contract with a microfluidic processorwhen inserted into the board. Such a cooler prevents the progressivebuild-up of the effects of heaters energized during the course of areaction or analysis. A cooling device may be controlled similarly to aresistive heater by adjusting a control current to maintain a specifictemperature sensor at the desired baseline temperature, where thespecific sensor is mounted at a thermal distance from heaters to sensebackground processor temperature.

Further Component-Level Functions

A further component-level function controllably generates pressure, forexample, to move micro-droplets or other materials in a microfluidicprocessor. This function is an important component of severalhigher-level actuators requiring thermally-controlled mechanical force.A preferred embodiment of a pressure generator includes a gas reservoirwith a controlled heating element and a passage conducting gaspressurized by heating to its point of application. FIG. 5A illustratesa preferred embodiment with relatively larger gas reservoir 65 andrelatively smaller conducting passage 66 linking to the point ofpressure application in passage 68. The gas in the reservoir ispreferably inert, such a nitrogen or argon, but can be air. Thereservoir has controlled heater 69 (the accompanying temperature sensoris not illustrated) embedded in its base (or top). Region 67 of passage68 has a hydrophobic surface so that any (aqueous) fluid present inpassage 68 is excluded from gas reservoir 69.

FIG. 5B illustrates a component-level control function for this pressuregenerator. In the first step, the function obtains identification of thepressure generator and its associated heater and a parameterrepresenting the desired pressure to be generated. In a next step, thedesired pressure is converted into a desired quantity of heat needed,and in the final step, the heater is controlled (by a control signalacross connectors determined from component identity) to a temperaturefor a time sufficient to supply the needed heat.

In addition to micro-droplet sensors depending on temperature effectsdescribed previously, further component-level functions may controlother types of micro-droplet sensors present on a microfluidicprocessor. For example, micro-droplet sensors may be based on capacitivedetection, in which an impedance between two leads is altered by thepresence or absence of a micro-droplet. The DAQ board then includesswitchable impedance sensing circuits. Pressure sensors may also bepresent and can be used as micro-droplet position sensors as explainedsubsequently. Pressure sensors may also provide direct feedback for usein the controllable pressure function of FIGS. 5A-B.

Micro-Valve Function

A micro-valves function is an important actuator-level function thatwill be present in most microfluidic processors. FIG. 6A illustrates apreferred embodiment of a micro-valve for closing and opening controlledpassage 78. The micro-valve has a pressure generator, for example,including gas reservoir 75 with heater HTR1 and side passage 77connecting with controlled passage 78. Side passage 77 is blocked byplug 76 of low melting-point, inert material. The melting point ispreferably greater than the baseline operating temperature of themicrofluidic processor but less than the boiling point of anymicro-droplet controlled by this micro-valve in passage 78. For example,the melting point may be from 40° to 90° C., preferably from 50° to 70°C.; the material may be a wax (for example, an olefin) or a eutecticalloy (for example, a solder). The micro-valve also includes heater HTR2for controlled heating of side passage 77, and heater HTR3 forcontrolled heating of controlled passage 78, as illustrated. Sensorsoptionally accompanying these three heaters are omitted from FIG. 6A forsimplicity and without limitation.

The configuration of leads 79-82 is one arrangement that providesindependent control of all three heaters with only four directly-routedand non-overlapping control leads. This illustrated arrangement isexemplary. For example, six leads, two for each heater, may be providedinstead.

The micro-valve closing operation is described with reference to FIG.6A, which depicts the micro-valve in an opened state, FIG. 6B, whichdepicts the micro-valve in a closed state, and FIG. 6C, which depictsthe steps of the micro-valve close function. The close function firstobtains input parameters 83, which identify the particular micro-valveto be closed, its component heaters, and the connectors for the heatercontrol leads and for monitoring signals from any optional sensors. Theinput parameters also includes the current micro-valve state, which mustbe “open” for the micro-valve close function. (If the micro-valve isalready closed, the close function may simply exit). Next, step 84controllably heats HTR2 (by activating leads 79 and 80) and side passage77 to a temperature T₂ slightly, but sufficiently, above (for example,1° to 5° C. above) the melting temperature of plug 76 so that the plugmelts. After or simultaneous with plug melting, step 85 controllablyheats HTR1 (by activating leads 80 and 81) to a temperature T₁ and for atime so the sufficient gas pressure is generated to move the melted plugfrom exit passage 77 into controlled passage 78. Preferably, T₁>T₂. Thispressure is maintained for a time delay 86 determined to be sufficientfor the plug to move into passage 78. Alternatively, where a positionsensor for the plug is available (for example, a thermal sensor inassociation with HTR3), the delay lasts until sufficient movement of theplug is sensed.

Step 87 then deactivates HTR2 and waits until its temperature returnswithin tolerances to T₀, the baseline processor temperature, so that theplug solidifies again. The return to baseline temperature may either besensed by a sensor or may be assumed after sufficient time delay. Afterthe plug is solidified, step 88 similarly returns the temperature ofHTR1 and gas reservoir 75 to baseline. Because the volume of the gas isnow greater because of the motion of the plug out of passage 77, arelatively lower gas pressure is present in reservoir 75 at the baselinetemperature when the micro-valve is closed than when it is open.

The micro-valve is now closed because controlled passage 78 is blockedwith the solidified plug. Step 89 marks the state of the micro-valve asclosed in the data describing current microfluidic processorconfiguration.

The micro-valve opening operation is described with reference to FIG.6B, which depicts the micro-valve in a closed state, FIG. 6A, whichdepicts the micro-valve in an opened state, and FIG. 6D, which depictsthe steps of the micro-valve open function. As customary, this functionfirst obtains 90 input parameters. These parameters identify the controland monitoring connectors and indicate a closed stated (otherwise, thefunction simply exits). First, the function controllably heats HTR2 andside passage 77 to temperature T₂ and HTR3 and controlled passage 78 totemperature T₁. T₁ and T₂ are both above the melting point of the plug,as described above. Plug 76 in controlled passage 78 thereby melts, and,under the influence of the relatively lower pressure in gas reservoir 75remaining from the micro-valve closing, is drawn back into side passage77. These heaters are activated for a time delay 91 determined to besufficient for the plug to move back into side passage 77.Alternatively, where a position sensor for the plug is available (forexample, a thermal sensor in association with HTR2), the delay is untilmovement of the plug is sensed. Finally, heaters HTR2 and HTR3 aredeactivated, and step 92 waits until the temperature in the vicinity ofthe exit passage heater has returned to within tolerances to baseline(either by temperature monitoring or by time delay). Finally, themicro-valve state is marked as closed with the plug now solidified inexit passage 77 and controlled passage 78 unblocked.

In the following descriptions, for ease of illustration and withoutlimitation, micro-valves are schematically represented with a singleheater and a single pair of leads, instead of their full illustration,as in FIGS. 6A-B, with three heaters and at least four leads.

Optical Detection Function

Optical sensing of the results of microfluidic processor reactions oranalyses is preferred because it may be easily performed externally to amicrofluidic-processor without any physical removal of reaction resultsfrom these passages. Alternatively, where a microfluidic processorincludes a separation facility for reaction results, detection ofcomponents separated thereby is also preferably by optical means.Optical sensing may depend on scattered incident radiation or generatedfluorescent radiation, or so forth. The invention also provides for theexcitation of a reaction or analysis by radiation.

Basic optical detection components and control functions are illustratedwith reference to FIGS. 11A-B. FIG. 11A illustrates limited section 165of a microfluidic processor with exemplary components for optic sensingof micro-droplet md1, which is illustrated as stablely positionedadjacent to hydrophobic region h1 of main passage 167. Optic componentsinclude radiation conductor 166 for conducting incident radiation (forexample, from a DAQ-board laser diode) to md1, and radiation conductor169 for conducting radiation from micro-droplet 1 for analysis (forexample, to a DAQ-board photodiode). Radiation conducted from md1 may bescattered radiation, fluorescent radiation, or so forth. Lens 168schematically illustrates elements for radiation gathering or focusing,filtering wavelengths, or so forth, present on the processor. Also, areflector may be placed adjacent to the main passage to double theradiation path through the micro-droplet being sensed. Such a reflectormay optionally have wavelength-dependent properties, being, for example,an interference filter or a dichroic mirror.

Limited portion 165 could be a substantially vertical depiction,illustrating substantially vertically arranged optic conductors out ofthe plane of the microfluidic processor and passing illumination throughthe thickness of the processor. This portion could also be asubstantially horizontal depiction, illustrating substantiallyhorizontally arranged optic conductors in the plane of the microfluidicprocessor and passing illumination through only one passage of theprocessor. Further, the optic conductors may run substantially in theplane of the processor (horizontally), only to angle to a finalorientation near their target.

FIG. 11B illustrates an actuator-level, optical-sensing function. Thisfunction begins by obtaining parameters 170 that identity the particularoptical-sensing actuator, so that the DAQ-board microprocessor maycontrol those radiation generation and detection components that connectto the correct connectors for the identified detection optical sensor.Next, the input radiation conductor is illuminated 171, and theresulting radiation is sensed 172.

Other such entirely external detection methods, based onexteriorly-applied magnetic (NMR) or electric fields, or on acombination of these fields with optical detection, can also bepreferably used in the microfluidic processors of this invention. Inthis case, field generation components must be placed on a microfluidicprocessor or on the DAQ board (or on a DAQ-board housing), must beidentified to the control function, and must be activated by the controlfunction.

Micro-Droplet Move Function

A micro-droplet move function is an important configuration-levelfunction that will be present in most microfluidic processors. Thisfunction moves a micro-droplet from a first position to a secondposition, thereby advancing the microfluidic processor from a firstconfiguration to a second configuration in which the micro-droplet movedis in its second position. This, and other micro-droplet-levelfunctions, act most reliably when the initial and final micro-dropletpositions are stable. They are preferably only invoked or only act whenthe data describing the current processor configuration indicates thatmicro-droplets are corrected positioned at a stable position. Asdescribed, a stable position can be established by, for example, ahydrophobic region in a passage, or by a local configuration ofpassages.

Micro-droplet motion of course requires thermally-controlled mechanicalforce and generally, in preferred microfluidic processors, thismechanical force is gas pressure-generated by a pressure generatoractuator. Micro-droplet motion may be stopped when the motion pressureis dissipated by, for example, a vent to the processor exterior. Motionmay also be stopped by a hydrophobic region which requires more motiveforce than is being supplied (coupled with deactivating the pressuregenerator actuator).

Micro-droplet motion is illustrated with reference to FIG. 7A, depictingmicro-droplet 95 in initial position 96, FIG. 7B, depictingmicro-droplet 95 in final position 97, and to FIG. 7C, depicting thesteps of the micro-droplet-motion function. Micro-valves in thesefigures (and subsequently) are schematically represented with only asingle heater. Step 100 of the move function customarily obtainsparameters identifying the components of the sub-assembly for movingmicro-droplet 95, here, valve1, valve2, and HTR1, their spatialrelationship, and their control leads or external connectors. In theexemplary configuration illustrated, initial micro-droplet position 96is just beyond the side passage to vent 1, a stable position after aprior movement with valve1 open, leaving vent1 accessible from mainpassage 98 to dissipate any driving pressure. Alternatively (notillustrated), the initial stable position may be defined by ahydrophobic region in passage 98, and valve1, vent1, and theirconnecting side passage may be absent from the microfluidic processor.Micro-droplet 95 is determined from the input configuration data to becorrectly in an initial stable position 96, because the known, current,microfluidic-processor configuration records the position of allmicro-droplets present in the processor. If there is no micro-droplet atposition 96, there is nothing for this function to do, and it exits.Preferably, the present motion function is called by a higher-levelfunction only when micro-droplet 95 is in position 96 as a result ofprior functions.

Next, step 101 prepares the micro-valves for micro-droplet motion byinvoking the actuator-level micro-valve functions to close valve1 (ifpresent and previously open, as determined by its state in themicrofluidic processor configuration data) and to open valve2 (ifpreviously closed, as also determined by its state). Step 102 thengenerates a pre-determined pressure by invoking actuator-levelpressure-generation functions 102. The generated pressure moves themicro-droplet to the right (it being assumed that generated pressure isnot dissipated to the left in passage 98 and that passage 98 is “open”to the right of position 97), until final position 97 where the appliedpressure dissipates to the exterior through vent2. The micro-dropletitself is prevented from entering vent2 by the hydrophobic protection ofits connecting side passage. When the micro-droplet reaches its finalposition, after delay 103, step 104 halts the pressure generationfunctions by deactivating HTR1. The duration of active pressuregeneration may be determined as a pre-selected time interval, optionallydependent on the processor configuration. Alternatively, where amicro-droplet position sensor is available (for example, a thermal-typesensor or a capacitive-type sensor), step 103 may wait until themicro-droplet is sensed to be in the final position. Optional step 105invokes the micro-valve functions to return valve 1 and valve2 to theirstates before micro-droplet motion.

Finally, upon successful completion, the micro-droplet position in themicrofluidic-processor configuration data is then updated in step 106.

Micro-Droplet Metering Function

The configuration-level micro-droplet metering function creates a newmicro-droplet of a more precisely known and smaller volume of fluid froma usually less precisely known and larger fluid volume (a reactant, asample, or so forth) introduced into a microfluidic processor.

Fluids may be introduced into a processor through ports by manualtransfer means (for example, a pipette) or by automatic transfer means(for example, a robot) from an exterior source. Ports may be provided onthe microfluidic processor to accept various fluid transfer means, forexample syringes or pipettes. FIG. 8C illustrates an exemplary portadapted for syringes. Port 1 includes fluid reservoir 114, covered withpuncturable membrane 113 (for example, of a self-sealing, rubber-likematerial), and connected to passage 110 in a microfluidic processor.This figure illustrates syringe 115 having punctured membrane 113 andalready having introduced fluid into the port. The membrane insuresinjected fluid penetrates into the processor without back flow. In thecase of pipettes, the shape of reservoir 114 may be adapted to a sealedfit with the pipette tip for fluid transfer.

In a preferred embodiment, a new micro-droplet is metered by beingpinched off from the larger volume, generally by means of a gas pressureforce. Micro-droplet metering is illustrated with reference to FIG. 8A,which shows an initial configuration before metering, FIG. 8B, whichshows a final configuration with new, metered micro-droplet 112, and toFIG. 8C, which shows steps of the preferred metering control function.FIG. 8A illustrates fluid aliquot 111, having been introduced throughport1 (such as the port illustrated in FIG. 8C) filling passage 110 upto the stable position formed by hydrophobic region h1. Hydrophobicregion h2 prevents fluid entry into the side passage to HTR1. Excessfluid may escape through vent 1, since valve1 is initially open, andexcess gas may escape though vent2, since valve2 is initially open.Passage 110 is designed, i.e., by having the illustrated relative sizes,so that fluid aliquot 111 experiences greater capillary force there thanin the side passage to vent1, in order that the fluid aliquot extends tothe hydrophobic patch before excess liquid extends to vent1. Thisconfiguration of passage sizes further stabilizes the stable positionformed by hydrophobic region h1.

The metering operation begins, as usual, at step 120, which identifiesthe metering components, their states, their arrangement, order, andtheir signal lines or external connectors. Optional step 121 opensvalve1 and valve2 by means of the actuator-level micro-valve functions,if they were not initially open. Next, the metering function waits 122for the loading of the fluid aliquot from which a micro-droplet is to bemetered. Its loading may be indicated by an external manual signalprovided to the user equipment (and transmitted to the DAQ board), ormay be automatically indicated by completion of robotic loading, or maybe provided by an internal sensor can detect the presence of fluidadjacent to hydrophobic region hi of passage 110. Step 123 then closesvalve1 by invoking the micro-valve close function, so that no more fluidmay escape out of vent 1.

Step 124 generates pressure by invoking the actuator-level pressuregenerator function (which activates HTR1). The pressure generator iscontrolled to a pre-determined pressure (if pressure sensors areavailable) or, alternatively, to a pre-determined metering temperatureThe resulting gas pressure pinches a length L of aliquot 111 that liesbetween the exit of the side passage to the pressure generator and theend of the aliquot at the stable position, forming a new micro-droplet.The volume of the metered micro-droplet is determined by length L andthe cross-section of passage 110. With reference now to FIG. 8B, thegenerated pressure further acts to move new micro-droplet 112, in themanner of the micro-droplet motion function described above, to position117, which is just beyond a side passage to vent2. The generatedpressures dissipates out vent2 since valve2 is open. Steps 125 and 126cease pressure generation after a pre-determined delay, or alternativelyafter micro-droplet 112 is sensed to be in position 117 (by amicro-droplet position sensor). Finally, an optional step closes valve2,to prevent further gas escape, thereby keeping the new micro-dropletfrom rejoining fluid aliquot 111. Valve1 may returned to its initialstate.

Lastly, step 127 updates the microfluidic processor configuration toreflect the presence, location, and composition (the same as aliquot111) of the new micro-droplet.

Micro-Droplet Mixing Function

Effective mixing of inhomogeneous micro-droplets is useful becausesimple diffusion, especially of biological macromolecules, is often tooslow to be practicable, even for adjacent micro-droplets in physicalcontact. Generally, micro-droplet mixing is achieved by motion that issufficiently rapid, in view of the passage size and droplet viscosity,to induce micro-droplet mixing. Preferably, the micro-droplet velocityequals or exceeds the critical inter-layering velocity. In a preferredembodiment, a micro-droplet-level mixing function may invoke amicro-droplet-level motion function in such a manner that the motion issufficiently rapid. This may be achieved by activating the pressuregenerator actuator, which provides the mechanical force to move themicro-droplet, so that the generated pressure rises sufficiently rapidlyto a sufficiently high level to cause rapid motion. Appropriateactivation of the pressure generator heater so that mixing ofmicro-droplets of particular viscosities occurs in passages of varioussizes can be readily be determined experimentally and stored for use bythe mixing function.

Inhomogeneous micro-droplets requiring mixing may arise for variousreasons. For example, FIGS. 9A-C illustrate formation of aninhomogeneous micro-droplet as a result of metering two different fluidaliquots into two adjacent micro-droplets. FIG. 9A illustrates portionsof two metering assemblies, metering1 and metering2, after loadingaliquot 131 of a first fluid and aliquot 132 of a second fluid, butprior to micro-droplet metering. (FIGS. 8A-B illustrate such meteringassemblies in full.) Pressure generator heaters, HTR1 and HTR2, areparts of these two metering assemblies. FIG. 9B next illustratesmicro-droplet, md1, in position 133 after it has been metered fromaliquot 131. Next, FIG. 9C illustrates md2 in position 133 after it inturn has been metered from aliquot 132. Md2 is positioned adjacent tomd1, and these two micro-droplets now form, in effect, a singleinhomogeneous micro-droplet.

The mixing function is now described with reference to FIG. 9C, whichdepicts the configuration prior to mixing, FIG. 9D, which depicts theconfiguration after results of mixing, and FIG. 9E. This latter figuredepicts the described preliminary metering steps 135 and 136 whichprepare an inhomogeneous micro-droplet for mixing, as well as steps 137of the actual mixing function. Step 138, as customary, obtains necessaryinput parameters, including identification of the mixing assemblycomponents (here, portions of the two metering assemblies) and theircontrol leads, and positions of the micro-droplet to be mixed. In thiscase, pressure for micro-droplet mixing may be generated by either orboth of the pressure generators present in the metering components. Step139 invokes actuator-level micro-valve functions to close valve1 (whichis usually open as a result of the previous metering steps), and to openvalve2, if necessary. Next, step 140 invokes the actuator-level pressuregeneration function to rapidly generate pressure, using either or bothof HTR1 and HTR2 heated to a sufficient temperature (the mixingtemperature) to cause mixing of the micro-droplet. Step 141 delays until(or senses when) micro-droplet md3 has reached position 134, then step142 closes valve2 behind the md3.

Lastly, step 144 updates the microfluidic processor configuration datato reflect the location and composition (now mixed) of the newmicro-droplet.

Perform Reaction Function

Generally, a micro-droplet that has been created with the correctcomposition is ready for the intended reaction or analysis. Preferably,for reaction, this micro-droplet is then isolated in order to avoidevaporation or unintended interactions with the remainder of themicrofluidic processor, and adjusted to a determined temperature, inorder that the reaction proceeds as intended. Certain reactions, notablythe polymerase chain reaction (PCR), may require that the micro-dropletbe repeatedly cycled through a determined temperature protocol. Otherreactions may require a (solid) catalyst, which will need to be in thereaction region of the microfluidic processor. Further, reactions mayrequire radiation stimulation. Although the following description is,without limitation, in terms of reactions at a determined temperature,one of average skill in terms of the following description will readilyunderstand how to provide for temperature protocols, catalysis,radiation stimulation and so forth.

Therefore, in the preferred embodiment described, reactions areperformed in a controllably-heated region of a passage that may beisolated from the rest of the microfluidic processor, or in acontrollably-heated reservoir into which a micro-droplet can be movedand isolated. FIG. 10A illustrates exemplary reaction region 156(without any catalyst) in passage 150, having a controllable heater,HTR1, and isolating valves, valve1 and valve2. Region 156 is a stableposition for a micro-droplet because of, for example, side passage 151leading to a controllable vent. (Similar stable positions are discussedwith respect to, inter alia, FIG. 1.) Alternately, a suitably placedhydrophobic region may define this stable position.

A reaction control function is illustrated with respect to FIG. 10C,which depicts reaction region 156 before reaction, to FIG. 10D, whichdepicts reaction region in the course of the reaction, and to FIG. 10E,which depicts the steps of a reaction control function. Step 157 obtainsparameters including microfluidic processor configuration, thetemperature profile for the reaction, the identities of the componentsforming the reaction region and their control leads and connectors. Fromthe-obtained configuration, this function checks that a micro-droplethaving the correct composition is positioned in the reaction region as aresult or prior microfluidic processing steps. If not, this functionexits, perhaps with an error indication. Next, step 158 invokes theactuator-level micro-valve functions to isolate reaction region 156 byclosing valve1 and valve2. Step 159 performs the prescribed thermalprotocol. Since no micro-droplet positions are changed by this function,the configuration need be updated only to the extent of indicating thata reaction has been performed.

This reaction completed may be the final result of the microfluidicprocessing, in which case the contents of the resulting micro-dropletare sensed, or it may be an intermediary reaction, in which case theresulting micro-droplet is further processed.

Next, exemplary construction of preferred, thermally-controlledmicrofluidic processors is briefly described. For example, FIG. 10Billustrates a section of FIG. 10A along line 10A-10B depicting generalconstruction of such processors from top plate 152, and parallel bottomplate 155, which is positioned and bonded with a seal against the topplate. The plates may be silicon, plastic polymer, glass or so forth.Passages, such as passage 150, are machined, etched, pressed, orotherwise defined in one plate, here the top plate, while the bottomplate is substantially flat, and have walls appropriately treated forthe type of micro-droplets to be processed. In particular, hydrophobic(or hydrophilic) passage regions are defined by passage treatmentsbefore plate bonding. Electrical components and leads, such as lead 154,are deposited preferably on the non-etched, substantially flat plate,and are covered (also, underlain if necessary) by insulating layer 153,which is inert to contents of the passages. Leads may be vapor depositedmetal, for example, aluminum. Insulating layer may be a ceramic orpolymer, for example, silicon dioxide. Light conductors may be made fromoptic fibers attached to a processor after plate bonding. Constructionmethods are adapted from those well known in the lithographic arts usedin semiconductor fabrication. See, for example, U.S. Pat. Nos.6,048,734, 6,057,149, and 6,130,098.

Integrated Device Operation Functions

The previously described micro-droplet-level functions can be combinedto create user-level reaction-control functions for many different typesof microfluidic processors performing many different reactions oranalyses. Construction, or programming, of such control functionsaccording to the present invention is enormously simplified becauseattention need generally only be paid to intuitive micro-droplet-levelfunctions, which specify laboratory functions familiar to chemists andbiologists, such as metering, moving, mixing, or reacting. Details ofindividual microfluidic-processor components and of their sequentialcontrol are hidden by the hierarchical construction of thecomponent-level, actuator-level, and micro-droplet-level controlfunctions, all of which function cooperatively to perform necessarylow-level microfluidic processor control. This hierarchical control ispossible because of the digital nature of the controlled microfluidicprocessors.

These advantages are illustrated by a user-level reaction-controlfunction for the preferred thermally-controlled microfluidic processorillustrated in FIG. 1. This processor is capable of performing, interalia, a simple PCR analysis of sample by metering a first micro-dropletcontaining the sample and some PCR reagents, by metering a secondmicro-droplet containing remaining PCR reagents, by mixing the twomicro-droplets, by performing a PCR temperature protocol on the mixedmicro-droplet, and by detecting the reaction results.

In more detail, FIG. 12 illustrates a user-level PCR-reaction-controlfunction, controlled by user commands entered on a host system. Step 175starts the reaction-control function after a user enters a command athost equipment. Next, steps 177 and 178 obtains input parameters, whichinclude data descriptive of the microfluidic processor on which thereaction is to be performed. As described, this descriptive data may beprovided by the microfluidic processor itself, or the processor mayprovide a key to a database of such data. This function identifies thecomponents, actuators, and sub-assemblies required by subsequentmicro-droplet-level functions, and preferably checks that this processorhas these correct resources in a correct arrangement. In themicrofluidic processor of FIG. 1, these resources are checked andidentified as metering1, metering2, mixing1, and reaction/detection1.Next, steps 177 and 178, using the metering micro-droplet-levelfunctions parameterized by the metering1 and metering2 sub-assemblies,meter first and second micro-droplets, including a sample for PCRanalysis and reagents. Both metering steps wait (see, for example, FIG.8D) for a signal indicating the aliquots from which the micro-dropletsare metered have been loaded into the processor. Next, step 179 invokesthe mixing micro-droplet-level function parameterized by the mixing1sub-assembly to mix the metered micro-droplets. Since the mixedmicro-droplet is now located in the reaction region of thereaction/detection1 sub-assembly, step 180 performs the reaction byinvoking the perform-reaction micro-droplet-level function. Lastly, step181 optically analyzes the reaction results by invoking the sensereaction results actuator-level function. Upon reaction completion, step182 returns the reaction results and a completion signal to the hostsystem. Throughout the operation of this function, asynchronous hostmonitoring or control 183 may be in progress, for example, by monitoringthe microfluidic processor configuration data as it is updated by thevarious invoked functions.

Therefore, this exemplary PCR reaction can be specified entirely interms of high-level micro-droplet functions. Detailed operations ofseveral individual components that must be coordinated to perform thisfunction are generally encapsulated by the micro-droplet functions inwhich the reaction control is expressed.

In an alternative embodiment, the reaction control function, afterobtaining the microfluidic processor description, determines itselfwhich processor components to use to perform the intended reaction. Ifmicro-droplets need to be moved between components that are not directlyconnected, the control function may insert the necessary micro-dropletmove function invocations. This determination is analogous to the layoutand wiring of a hardware description expressed in high-level hardwaredescription language (such as VHDL) on a semiconductor chip, and can beperformed by similar methods. Further alternatives apparent to one ofskill in the art are also included in this invention.

Sample Preparation

The control systems and methods of the present invention areadvantageously applied to control microfluidic processors to performpre-determined analyses of biological and medical samples. Exemplaryanalyses include determining the presence of certain nucleic acids orproteins that may indicate a disease state of an organism and help indiagnosing the disease state.

Accordingly, FIG. 13 illustrates the preparation of such samples foranalyses. First, a biological or medical specimen is obtained, such assamples obtained from the exterior of an organism, for example, byscraping or swabbing, or from the interior of an organism, for example,by biopsy or surgical specimen. Next a sample is prepared from thespecimen. This may include the steps of purifying the specimen fromextraneous material (removing cells where extracellular material is tobe analyzed), lysing cell (where intracellular materials are to beanalyzed), separating the type of material to be analyzed from othertypes (for example, nucleic acids from proteins). Finally, the preparedsample is loaded into a microfluidic processor for analysis by thesystems and methods of this invention.

The invention described and claimed herein is not to be limited in scopeby the preferred embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

A number of references are cited herein, the entire disclosures of whichare incorporated herein, in their entirety, by reference for allpurposes. Further, none of these references, regardless of howcharacterized above, is admitted as prior to the invention of thesubject matter claimed herein.

1. A system for controlling the operation of a microfluidic devicehaving a micropassage configured for holding a micro-volume of liquidhaving a volume between one nano-liter and one micro-liter, a reactionchamber, and one or more active components, the system comprising: aprocessor configured for receiving a user request for the microfluidicdevice to perform a reaction program; memory comprising storedinstructions corresponding to hierarchical control signals configured todirect the microfluidic device to perform the user-requested reactionprogram; and a programmable digital acquisition unit comprising: aheater driver circuit; a temperature sensor driver circuit; and, adetection driver circuit wherein the detection driver passes signals toat least one active component configured to detect reaction products inthe reaction chamber; wherein the digital acquisition unit generatescontrol signals for the active components responsive to theuser-requested reaction program; wherein the control signals control (i)heating the micro-volume of liquid, (ii) detecting a temperature relatedto the micro-volume of liquid and (iii) detecting reaction products inthe micro-volume of liquid.
 2. A system for controlling the operation ofa microfluidic device as recited in claim 1, further comprising at leastone receptacle for receiving the microfluidic device, wherein thereceptacle provides for transfer of the control signals between theprogrammable digital acquisition unit and the microfluidic device.
 3. Adetection driver circuit as recited in claim 1, further comprising alight emitter driving circuit and a light detector driving circuit.
 4. Asystem for controlling the operation of a microfluidic device as recitedin claim 1, wherein the hierarchical control comprises a user levelfunction associated with the user-requested reaction program.
 5. Asystem for controlling the operation of a microfluidic device as recitedin claim 4, wherein the hierarchical control comprises a microdropletlevel function corresponding to an operation performed on a micro-volumeof liquid contained within a microfluidic device.
 6. A system forcontrolling the operation of a microfluidic device as recited in claim5, comprising an actuator comprising associated and active componentsconfigured for coordinated operation to achieve a desired functionality7. A system for controlling the operation of a microfluidic device asrecited in claim 6, wherein the hierarchical control comprises anactuator level function corresponding to an actuator operation.
 8. Asystem for controlling the operation of a microfluidic device as recitedin claim 7, wherein the actuator level function comprises instructionsto detect the presence or absence of the micro-volume of liquid.
 9. Asystem for controlling the operation of a microfluidic device as recitedin claim 8, wherein the hierarchical control comprises a component levelinstruction directing the generation of a control signal for anindividual component of the microfluidic device.
 10. A system forcontrolling the operation of a microfluidic device as recited in claim9, wherein the hierarchical control provides instructions to themicrofluidic device to (i) move the micro-volume of liquid to thereaction chamber, (ii) close a valve, (iii) perform thermal cyclingwithin the reaction chamber, and (iv) detect reaction products in thereaction chamber.
 11. A system configured for controlling a microfluidicdevice, wherein the microfluidic device comprises a micro-channelconfigured to contain a micro-volume of liquid having a volume betweenone nano-liter and one micro-liter, a reaction chamber, and an actuatorcomprising associated and active components configured for coordinatedoperation to achieve a desired functionality, the system comprising:memory comprising stored instructions corresponding to a user-selectedreaction program, wherein the stored instructions comprise: a user levelfunction corresponding to the reaction program; a microdroplet levelfunction corresponding to an operation performed on a micro-volume ofliquid contained within a microfluidic device; an actuator levelfunction corresponding to an actuator operation; and, a component levelinstruction directing the generation of a control signal for anindividual component of the microfluidic device; an interface configuredfor allowing an operator to select a desired reaction program for themicrofluidic device, wherein the desired reaction program corresponds toa user level function comprising a microdroplet level function; whereinthe microdroplet level function comprises an actuator level function,and the actuator level function comprises a component level function;and, control circuitry configured for creating and transmitting thecontrol signal responsive to a component level function for controllingthe component of the microfluidic device.
 12. The system for controllinga microfluidic device as recited in claim 11, further comprising atleast one receptacle for receiving the microfluidic device, wherein thereceptacle provides for transfer of the control signals between thecontrol circuitry and the microfluidic device.
 13. The system forcontrolling a microfluidic device as recited in claim 11, wherein one ofthe active components comprises a light emitter and one of the activecomponents comprises a light detector.
 14. The system for controlling amicrofluidic device as recited in claim 11, further comprising storedinstructions directing a laboratory robot to introduce a micro-volume ofliquid into the microfluidic device.
 15. The system for controlling amicrofluidic device as recited in claim 11, wherein the hierarchicalcontrol provides instructions to the microfluidic device to (i) move themicro-volume of liquid to the reaction chamber, (ii) close a valve,(iii) perform thermal cycling within the reaction chamber, and (iv)detect reaction products in the reaction chamber.
 16. The system forcontrolling a microfluidic device as recited in claim 15, whereinreaction products in the reaction chamber are detected by measuringfluorescence of the contents of the reaction chamber.
 17. The system forcontrolling a microfluidic device as recited in claim 16, wherein thehierarchical control actuates a light source to illuminate a portion ofthe reaction chamber.
 18. The system for controlling a microfluidicdevice as recited in claim 16, wherein the hierarchical control actuatesa light detector to detect fluorescent emissions from the contents ofthe reaction chamber.
 19. The system for controlling a microfluidicdevice as recited in claim 11, wherein the actuator level functioncomprises instructions to detect the presence or absence of themicro-volume of liquid.
 20. The system for controlling a microfluidicdevice as recited in claim 19, wherein the hierarchical control includesinstructions to determine the local heat capacity of a portion of themicrofluidic device to determine the presence or absence of themicro-volume of liquid.